Bridges Lab Protocols - User contributions [en]

Safety and Animal Training

2022-10-26T19:39:37Z

Mollyec: updated courses to include MiChart

These are the classes that lab staff need to take at Michigan:

==General Lab Safety==
These are offered through MyLink at https://maislinc.umich.edu/. All lab personnel need to take these

* EHS_BLS025w ONLINE REQUIRED General Laboratory Safety Training (online)
* BLS023 REQUIRED Centrifuge Training (classroom)
* BLS013w ONLINE REQUIRED Autoclave Standard Operating Procedures (online)

If you will be working with blood you will need to take

* BLS101w ONLINE REQUIRED Bloodborne Pathogens (online)

If you will be working with radioactive materials, you will have to take this course as well:

* RSS006 REQUIRED Radiation Safety Orientation (classroom)
* RSS103w REQUIRED ONLINE REQUIRED Radionuclide Users Annual Refresher Training (to be completed every CALENDAR year)
If you will be working with any viruses you will have to take this course as well:

* BLS008 REQUIRED Working Safely with Viral Vectors (classroom)

If you plan to breed animals, you can also take this course:

*ULAM-10125 Breeding Colony Management for Rats and Mice (online)

==Animals==
If you will be working with animals you will have to take these classes. They are all offered through ULAM MLearning (https://trainingportal.med.umich.edu/). This requires a levels 2 password.

* ULAM-10000 Orientation (online)
* ULAM-10050 Hazards pt.1 (online)
* ULAM-10055 Hazards pt.2 (classroom)
* ULAM-10100 Intro Mouse/Rat (online)
* ULAM-10131 Animal Room Procedures pt.1 (online)
* ULAM-10132 Animal Room Procedures pt.2 (classroom)
* ULAM-10105 Lab Mouse Workshop (classroom)

==Human Subject Data==
If you will be working with Human data you will have to take these classes. They are all offered through MLearning (https://trainingportal.med.umich.edu/). This requires a levels 2 password.

* PRIV-10001 HIPAA Training for All Workforce Members (online)
* PEERRS (online)
* DCE101 U-M Data Protection and Responsible Use (online, for HSIP)
* MiChart Research Access eLearning Research Certification (MC-RE20001V)(Online)
* MiChart Inpatient Allied Health HOD Certification (MC-HD70003A)(Online)

==Lab Standard Operating Procedures==
For lab specific safety training please review the SOP's and sign them if you will be using any of them. All lab SOP's are available [[:Category: SOP|here]]

[[ Category: Lab Safety ]]
[[ Category: Training ]]
[[Category: SOP]]

Rate of drop analysis

2022-09-01T13:54:39Z

Mollyec:

==Purpose==
To assess the initial response to insulin administration, you can calculate the slope of the line for the early timepoints in your insulin tolerance test. To do this, complete the following steps:

==Methods==
1. First, filter your ITT data to contain only the observations from where animals are in the initial drop of the experiment. This is usually 30-45 minutes, but you should assess this for each ITT yourself.

2. You then need to construct a model for your data. It should include 1 covariate for every experimental variable (eg, time, genotype, sex, diet, injection). You should also include an interaction variable for time and the variable of interest. This will be the crucial value that you interpret different slopes of the initial drop from.

3. After creating the model, you will need to assess the significance of that model. Do this using the anova command in R. This will give you the covariate p values.

4. Next, to find the beta estimates, you will need to run the coefficient command in R. This will tell you the actual changes in glucose (mg/dL) for each covariate. The beta coefficient of interest is the one corresponding to the interaction variable you coded in step 2.

==Sample R Code==
models<-ITT.raw.data%>%
filter(time<60)%>%
group_by(ID, sex, treatment)%>%
mutate(l.glucose = log(glucose))%>%
do(fitted.model= lm(l.glucose~ time, data =.))%>%
mutate(rate =coef(fitted.model)["time"],
max = coef(fitted.model)["(Intercept)"],
rsq = summary(fitted.model)$r.squared)%>%
mutate(max.exp = exp(max))%>%
mutate( slope= max.exp*rate)

summary.models<-models%>%
group_by(sex,treatment)%>%
summarise_at(.var ="slope", .funs = funs(mean, se))

ggplot(summary.models, aes(treatment, mean, fill = treatment))+
geom_col(aes(fill = treatment))+
facet_grid(.~sex)+
geom_errorbar(aes(ymin = mean-se, ymax = mean + se), width = 0.3)+
scale_fill_manual(values = color.scheme)+
labs(title = "Rate of Drop, ITT",y="mg/dL per minute")

rate.aov<-aov(slope ~ sex + treatment, data = models)
anova(rate.aov)%>%kable

Rate of drop analysis

2022-09-01T13:52:51Z

Mollyec:

==Purpose==
To assess the initial response to insulin administration, you can calculate the slope of the line for the early timepoints in your insulin tolerance test. To do this, complete the following steps:

==Methods==
1. First, filter your ITT data to contain only the observations from where animals are in the initial drop of the experiment. This is usually 30-45 minutes, but you should assess this for each ITT yourself.

2. You then need to construct a model for your data. It should include 1 covariate for every experimental variable (eg, time, genotype, sex, diet, injection). You should also include an interaction variable for time and the variable of interest. This will be the crucial value that you interpret different slopes of the initial drop from.

3. After creating the model, you will need to assess the significance of that model. Do this using the anova command in R. This will give you the covariate p values.

4. Next, to find the beta estimates, you will need to run the coefficient command in R. This will tell you the actual changes in glucose (mg/dL) for each covariate. The beta coefficient of interest is the one corresponding to the interaction variable you coded in step 2.

==Sample R Code==
new.data.set <-original.data.set%>%
filter(time=="0"| time == "15" | time == "30"| time =="45")

drop.data.lm <- lm (formula = ( glucose ~ time + treatment + time:treatment, data = new.data.set)

anova(drop.data.lm)

coefficients(drop.data.lm)

Safety and Animal Training

2022-06-27T18:50:34Z

Mollyec:

These are the classes that lab staff need to take at Michigan:

==General Lab Safety==
These are offered through MyLink at https://maislinc.umich.edu/. All lab personnel need to take these

* EHS_BLS025w ONLINE REQUIRED General Laboratory Safety Training (online)
* BLS023 REQUIRED Centrifuge Training (classroom)
* BLS013w ONLINE REQUIRED Autoclave Standard Operating Procedures (online)

If you will be working with blood you will need to take

* BLS101w ONLINE REQUIRED Bloodborne Pathogens (online)

If you will be working with radioactive materials, you will have to take this course as well:

* RSS006 REQUIRED Radiation Safety Orientation (classroom)
* RSS103w REQUIRED ONLINE REQUIRED Radionuclide Users Annual Refresher Training (to be completed every CALENDAR year)
If you will be working with any viruses you will have to take this course as well:

* BLS008 REQUIRED Working Safely with Viral Vectors (classroom)

If you plan to breed animals, you can also take this course:

*ULAM-10125 Breeding Colony Management for Rats and Mice (online)

==Animals==
If you will be working with animals you will have to take these classes. They are all offered through ULAM MLearning (https://trainingportal.med.umich.edu/). This requires a levels 2 password.

* ULAM-10000 Orientation (online)
* ULAM-10050 Hazards pt.1 (online)
* ULAM-10055 Hazards pt.2 (classroom)
* ULAM-10100 Intro Mouse/Rat (online)
* ULAM-10131 Animal Room Procedures pt.1 (online)
* ULAM-10132 Animal Room Procedures pt.2 (classroom)
* ULAM-10105 Lab Mouse Workshop (classroom)

==Human Subject Data==
If you will be working with Human data you will have to take these classes. They are all offered through MLearning (https://trainingportal.med.umich.edu/). This requires a levels 2 password.

* PRIV-10001 HIPAA Training for All Workforce Members (online)
* PEERRS (online)
* DCE101 U-M Data Protection and Responsible Use (online, for HSIP)

==Lab Standard Operating Procedures==
For lab specific safety training please review the SOP's and sign them if you will be using any of them. All lab SOP's are available [[:Category: SOP|here]]

[[ Category: Lab Safety ]]
[[ Category: Training ]]
[[Category: SOP]]

Safety and Animal Training

2022-06-27T18:19:01Z

Mollyec:

These are the classes that lab staff need to take at Michigan:

==General Lab Safety==
These are offered through MyLink at https://maislinc.umich.edu/. All lab personnel need to take these

* EHS_BLS025w ONLINE REQUIRED General Laboratory Safety Training (online)
* BLS023 REQUIRED Centrifuge Training (classroom)
* BLS013w ONLINE REQUIRED Autoclave Standard Operating Procedures (online)

If you will be working with blood you will need to take

* BLS101w ONLINE REQUIRED Bloodborne Pathogens (online)

If you will be working with radioactive materials, you will have to take this course as well:

* RSS006 REQUIRED Radiation Safety Orientation (classroom)
* RSS103w REQUIRED ONLINE REQUIRED Radionuclide Users Annual Refresher Training (to be completed every CALENDAR year)
If you will be working with any viruses you will have to take this course as well:

* BLS008 REQUIRED Working Safely with Viral Vectors (classroom)

If you plan to breed animals, you can also take this course:

*ULAM-10125 Breeding Colony Management for Rats and Mice (online)

==Animals==
If you will be working with animals you will have to take these classes. They are all offered through ULAM MLearning (https://trainingportal.med.umich.edu/). This requires a levels 2 password.

* ULAM-10000 Orientation (online)
* ULAM-10050 Hazards pt.1 (online)
* ULAM-10055 Hazards pt.2 (classroom)
* ULAM-10100 Intro Mouse/Rat (online)
* ULAM-10131 Animal Room Procedures pt.1 (online)
* ULAM-10132 Animal Room Procedures pt.2 (classroom)
* ULAM-10105 Lab Mouse Workshop (classroom)

==Human Subject Data==
If you will be working with Human data you will have to take these classes. They are all offered through MLearning (https://trainingportal.med.umich.edu/). This requires a levels 2 password.

* PRIV-10001 HIPAA Training for All Workforce Members (online)
* PEERRS (online)
DCE101 U-M Data Protection and Responsible Use (online, for HSIP)

==Lab Standard Operating Procedures==
For lab specific safety training please review the SOP's and sign them if you will be using any of them. All lab SOP's are available [[:Category: SOP|here]]

[[ Category: Lab Safety ]]
[[ Category: Training ]]
[[Category: SOP]]

Safety and Animal Training

2022-06-09T19:55:22Z

Mollyec:

These are the classes that lab staff need to take at Michigan:

==General Lab Safety==
These are offered through MyLink at https://maislinc.umich.edu/. All lab personnel need to take these

* EHS_BLS025w ONLINE REQUIRED General Laboratory Safety Training (online)
* BLS023 REQUIRED Centrifuge Training (classroom)
* BLS013w ONLINE REQUIRED Autoclave Standard Operating Procedures (online)

If you will be working with blood you will need to take

* BLS101w ONLINE REQUIRED Bloodborne Pathogens (online)

If you will be working with radioactive materials, you will have to take this course as well:

* RSS006 REQUIRED Radiation Safety Orientation (classroom)
* RSS103w REQUIRED ONLINE REQUIRED Radionuclide Users Annual Refresher Training (to be completed every CALENDAR year)
If you will be working with any viruses you will have to take this course as well:

* BLS008 REQUIRED Working Safely with Viral Vectors (classroom)

If you plan to breed animals, you can also take this course:

*ULAM-10125 Breeding Colony Management for Rats and Mice (online)

==Animals==
If you will be working with animals you will have to take these classes. They are all offered through ULAM MLearning (https://trainingportal.med.umich.edu/). This requires a levels 2 password.

* ULAM-10000 Orientation (online)
* ULAM-10050 Hazards pt.1 (online)
* ULAM-10055 Hazards pt.2 (classroom)
* ULAM-10100 Intro Mouse/Rat (online)
* ULAM-10131 Animal Room Procedures pt.1 (online)
* ULAM-10132 Animal Room Procedures pt.2 (classroom)
* ULAM-10105 Lab Mouse Workshop (classroom)

==Human Subject Data==
If you will be working with Human data you will have to take these classes. They are all offered through MLearning (https://trainingportal.med.umich.edu/). This requires a levels 2 password.

* PRIV-10001 HIPAA Training for All Workforce Members (online)
* PEERRS (online)

==Lab Standard Operating Procedures==
For lab specific safety training please review the SOP's and sign them if you will be using any of them. All lab SOP's are available [[:Category: SOP|here]]

[[ Category: Lab Safety ]]
[[ Category: Training ]]
[[Category: SOP]]

Safety and Animal Training

2022-06-09T19:54:59Z

Mollyec:

These are the classes that lab staff need to take at Michigan:

==General Lab Safety==
These are offered through MyLink at https://maislinc.umich.edu/. All lab personnel need to take these

* EHS_BLS025w ONLINE REQUIRED General Laboratory Safety Training (online)
* BLS023 REQUIRED Centrifuge Training (classroom)
* BLS013w ONLINE REQUIRED Autoclave Standard Operating Procedures (online)

If you will be working with blood you will need to take

* BLS101w ONLINE REQUIRED Bloodborne Pathogens (online)

If you will be working with radioactive materials, you will have to take this course as well:

* RSS006 REQUIRED Radiation Safety Orientation (classroom)
* RSS103w REQUIRED ONLINE REQUIRED Radionuclide Users Annual Refresher Training (to be completed every CALENDAR year)
If you will be working with any viruses you will have to take this course as well:

* BLS008 REQUIRED Working Safely with Viral Vectors (classroom)

If you plan to breed animals, you can also take this course:

*ULAM-10125 Breeding Colony Management for Rats and Mice (online)

==Animals==
If you will be working with animals you will have to take these classes. They are all offered through ULAM MLearning (https://trainingportal.med.umich.edu/). This requires a levels 2 password.

* ULAM-10000 Orientation (online)
* ULAM-10050 Hazards pt.1 (online)
* ULAM-10055 Hazards pt.2 (classroom)
* ULAM-10100 Intro Mouse/Rat (online)
* ULAM-10131 Animal Room Procedures pt.1 (online)
* ULAM-10132 Animal Room Procedures pt.2 (classroom)
* ULAM-10105 Lab Mouse Workshop (classroom)

==Human Subject Dataa==
If you will be working with Human data you will have to take these classes. They are all offered through ULAM MLearning (https://trainingportal.med.umich.edu/). This requires a levels 2 password.

* PRIV-10001 HIPAA Training for All Workforce Members (online)
* PEERRS (online)

==Lab Standard Operating Procedures==
For lab specific safety training please review the SOP's and sign them if you will be using any of them. All lab SOP's are available [[:Category: SOP|here]]

[[ Category: Lab Safety ]]
[[ Category: Training ]]
[[Category: SOP]]

SOP - Tamoxifen

2022-06-06T16:50:02Z

Mollyec:

[[ Category: SOP ]]
[[ Category: Lab Safety]]
'''Laboratory Director (LD) Approval is Required Prior to Performing this Procedure'''

=Description=
This standard operating procedure (SOP) outlines the handling and use of tamoxifen. Review this document and supply the information required in order to make it specific to your facility. In accordance with this document, laboratories should use appropriate controls, personal protective equipment, and disposal techniques when handling tamoxifen.

Tamoxifen is classified as a hazardous drug according to the National Institute for Occupational Safety and Health (NIOSH). Tamoxifen is a triphenyl ethylene derivative with marked anti-estrogen properties, which binds to estrogen receptors and, as a result, has proven useful in the treatment of breast cancer (antineoplastic). Tamoxifen citrate has been used to stimulate ovulation in infertility.

Synonyms: Nolvadex, Istubal, Valodex

=Potential Hazards=
• May cause cancer.

• May impair fertility and cause harm to the unborn child or breastfed babies.

• Accidental ingestion of the material may be harmful.

• May cause eye irritation.

• This product may be harmful if inhaled and cause respiratory tract irritation. Persons with impaired respiratory function, airway diseases, and conditions such as emphysema or chronic bronchitis, may incur further disability if excessive concentrations of particulate are inhaled.

• The material is not thought to be a skin irritant (as classified using animal models). But may be harmful if absorbed through the skin and may cause skin irritation; systemic effects may result following absorption. Entry into the blood-stream through, for example, cuts, abrasions or lesions, may produce systemic injury with harmful effects. Examine the skin prior to the use of the material and ensure that any external damage is suitably protected.

=Engineering Controls=
• Use a biological safety cabinet or chemical fume hood during reconstitution and dilution of this material.

• Syringes used for tamoxifen injection must be safety engineered (self-sheathing syringes, luer-lock syringes, etc.)

• Use with animals typically requires ABSL2 containment housing.

• Animals should be appropriately restrained and/or sedated prior to administering injections and other dosing methods.

• Laboratories and other spaces where handling of tamoxifen occurs must be equipped with an eyewash station.

=Work Practice Controls=
• Avoid inhalation.

• Good hygiene practice requires that exposure be kept to a minimum and that suitable PPE and control measures be used in an occupational setting.

• Decontamination should consist of surface cleaning with water and detergent followed by thorough rinsing. The use of detergent is recommended because there is no single accepted method of chemical deactivation for all agents involved.

• A plastic backed absorbent pad should be placed under the work area during the process. This should be changed at the end of each procedure or when a spill occurs.

=Personal Protective Equipment (PPE)=
Appropriate lab attire including long pants and closed toe shoes must be worn. Personal protective equipment includes a lab coat or disposable coveralls, eye protection and two pairs of disposable nitrile gloves. Gloves should be changed frequently and should be pulled up over sleeves if possible to reduce the likelihood of any exposed skin. Wash hands and arms immediately after working with the substance.

Note: Respirators are masks designed to protect the wearer from specific airborne hazards and are different from surgical masks, which protect the wearer only from splashes and are primarily intended to protect others from infectious aerosols exhaled by the wearer. Respirator use requires employee participation in the Respiratory Protection Program, which involves medical clearance and annual fit testing and training. Please be clear about use of surgical masks versus respirators. (Do NOT use the vague term “masks”.)

=Transportation and Storage=
• Keep containers tightly closed and in dry, well-ventilated area.

• Keep away from light.

• Avoid strong oxidizing agents.

• Tamoxifen is regulated by DOT as a Hazardous Material as well as IATA for Transport of Dangerous Goods.

=Waste Disposal=
All hazardous chemical waste should be placed in an appropriate container for disposal (pail or bottle). The container should be closed except when actively adding waste. The container must be located in the area where hazardous drugs are being used.

All items contaminated by hazardous drugs, including gloves, syringes, vials needles, animal bedding and solution containers should be disposed according to OSEH guidelines.

• Sharps – place needles, syringes with needles attached and other breakable items into appropriately labeled sharps containers.

• Empty stock vials, reagent bottles, etc. – triple rinse with copious amounts of water. Deface label with black magic marker or scraper. Place in a cardboard box for disposal.

Because most spent, unused and expired chemicals/materials are considered hazardous wastes, they must be properly disposed of. Do not dispose of chemical wastes by dumping them down a sink, flushing in a toilet or discarding in regular trash containers, unless authorized by OSEH Hazardous Materials Management (HMM). Contact OSEH-HMM at (734) 763-4568 for waste containers, labels, manifests, waste collection and for any questions regarding proper waste disposal. Also refer to OSEH’s Hazardous Waste webpage for more information.

=Exposures/Unintended Contact=

If the employee is in need of emergency medical attention, call 911 immediately.

==SWALLOWED==
• Rinse mouth with water.

• Consult a physician.

==EYE==
If this product comes in contact with the eyes:
• Wash out immediately with fresh running water for a minimum of 15 minutes.

• Ensure complete irrigation of the eye by keeping eyelids apart and away from eye and moving the eyelids by occasionally lifting the upper and lower lids.

• If pain persists or recurs seek medical attention.

• Removal of contact lenses after an eye injury should only be undertaken by skilled personnel.

==SKIN==
If skin contact occurs:
• Immediately remove all contaminated clothing, including footwear.

• Flush skin and hair with running water (and soap if available) for a minimum of 15 minutes.

• Seek medical attention in event of irritation.

==INHALED==
• Move person from contaminated area into fresh air.

• Move person into fresh air.

• Seek medical attention.

Contact OSEH for advice on symptoms of chemical exposure, or assistance in performing an exposure assessment.

Report all work related accidents, injuries, illnesses or exposures to Work Connections within 24 hours by completing and submitting the Illness and Injury Report Form. Follow the directions on the Work Connections website Forms Instructions to obtain proper medical treatment and follow-up.

Complete the OSEH Laboratory Incident and Near-Miss Report form.

===TREATMENT FACILITIES:===
U-M Occupational Health Services -- Campus Employees
Mon-Fri 7:30 am - 4:30 pm

After hours - go to UM Hospital Emergency Dept. – Urgent Care Clinic
C380 Med Inn building
1500 East Medical Center Drive, Ann Arbor (734) 764-8021

University Health Services -- University students (non-life threatening conditions)
Mon-Fri 8 am – 4:30 pm, Sat 9 am – 12 pm
Contact for current hours as they may vary
207 Fletcher Street, Ann Arbor (734) 764-8320

UMHS Emergency Department -- after clinic hours or on weekends
1500 East Medical Center Drive, Ann Arbor, (734) 936-6666

=Spill Procedure=
• When a spill occurs, personal safety should always come first.

• Alert and clear everyone in the immediate area where the spill occurred.

A minor (small) chemical spill is one that the laboratory staff is capable of handling safely without the assistance of safety and emergency personnel, i.e., (less than 1 Gallon or 3.5 Liters). A major/large chemical spill requires active assistance from emergency personnel.

Spill Response Steps:

===MINOR CHEMICAL SPILL===
• Alert people in immediate area of spill.

• Open outside windows, if possible.

• Use proper personal protective equipment (PPE) as indicated above.

• Avoid breathing vapors from spill.

• Confine spill to as small an area as possible.

• Do not wash spill down the drain.

• Use appropriate spill kits/sorbents to absorb spill. Collect contaminated materials and residues and place in container. For powdered chemicals sweep carefully to avoid generation of dust or, if appropriate, use moist sorbent pads or wet the powder with a suitable solvent and then wipe with a dry cloth. Contact OSEH-HMM (734) 763-4568 for proper disposal.

• Clean spill area with water.

===MAJOR CHEMICAL SPILL===
Report large chemical spills (greater than 1 Gallon or 3.5 Liters) in corridors or common areas, e.g., hallways, elevators, eating areas, rest rooms, offices, etc., to University of Michigan Division of Public Safety and Security (DPSS) at 911.
• Attend to injured or contaminated persons and remove them from exposure.

• Alert people in the laboratory to evacuate.

• Call University of Michigan Division of Public Safety and Security (DPSS) at 911 immediately for assistance.

• Close doors to affected area.

• Post warnings to keep people from entering the area.

• Have person available that has knowledge of incident and laboratory to assist emergency personnel.

Additional Spill Links:
• http://oseh.umich.edu/hazardous-waste/spill-response/

Report all emergencies, suspicious activity, injuries, spills, and fires to the University of Michigan Division of Public Safety and Security (DPSS) by calling 911 or texting 377911. Register with the University of Michigan Emergency Alert System via Wolverine Access.

=Training of Personnel=
All personnel are required to complete the General Laboratory Safety Training session (BLS025w or equivalent) via OSEH’s My LINC website. Furthermore, all personnel shall read and fully adhere to this SOP when handling tamoxifen.

=Certification=
I have read and understand the above SOP. I agree to contact my Supervisor or Lab manager if I plan to modify this procedure.

*[[User:Iharvey|Iharvey]] ([[User talk:Iharvey|talk]]) 20:56, 10 November 2016 (UTC)
*[[User:Elhabbal|Elhabbal]] ([[User talk:Elhabbal|talk]]) 22:23, 12 June 2017 (UTC)
*[[User:Pfeiferl|Pfeiferl]] ([[User talk:Pfeiferl|talk]]) 16:48, 20 June 2017 (UTC)
*[[User:Mollyec|Mollyec]] ([[User talk:Mollyec|talk]]) 16:50, 6 June 2022 (UTC)
*[[User:Reddj|Reddj]] ([[User talk:Reddj|talk]])

Laboratory Director Revision Date
�
Major Revisions (Tracking purposes only – Do not print as part

SOP - Rapamycin

2022-06-06T16:49:16Z

Mollyec:

[[ Category: SOP ]]
[[ Category: Lab Safety ]]

==Description==
''This standard operating procedure outlines the handling and use of rapamycin. Review this document and supply the information required in order to make it specific to your facility. In accordance with this document, laboratories should use appropriate controls, personal protective equipment, and disposal techniques when handling rapamycin.''

Rapamycin is an immunosuppressant drug used to prevent rejection in organ transplantation; it is especially useful in kidney transplants. Rapamycin is not a calcineurin inhibitor. However, it has a similar suppressive effect on the immune system. Rapamycin inhibits the response to interleukin-2 (IL-2) and thereby blocks activation of T- and B-cells. The health effects from long-term usage could include increase the risk of developing an infection or cancer, especially lymphoma (cancer of a part of the immune system) or skin cancer.

Synonyms: RAPA, RPM, Rapamune, Sirolimus, "macrocyclic polyketide", "macrocyclic triene antibiotic", antirestenotic, immunosuppressant, "mTOR inhibitor"

==Potential Hazards==
*Toxic: danger of serious damage to health by prolonged exposure if swallowed.
*Possible carcinogenic effect.
*Biologically active compounds may produce idiosyncratic effects which are entirely unpredictable on the basis of literature searches and prior clinical experience (both recent and past).
*Ingestion of the material may be damaging to the health of the individual.

Macrolides comprise a large group of antibiotics derived from Streptomyces spp. having in common a macrocyclic lactone ring to which one or more sugars are attached. They are all weak bases. The most common side effect produced by the family of macrolide antibiotics is gastrointestinal discomfort. Supra-infections may occur although these are rare. Several macrolides produced allergic sensitization but, again, these are rare. Symptoms include watery eyes, shortness of breath, nasal congestion, choking, coughing and wheezing. Allergic skin reactions have also occurred. Exposure to at least one member of the family, erythromycin, at high concentrations, has produced reversible deafness (ototoxicity). Systemic reactions including fever, rash, and lymph-node pain or swelling have been produced by the avermectin group. Ivermectin has produced ataxia (incoordination), lethargy, bradypnea (slowed breathing), vomiting, mydriasis (dilated pupils), sedation, tremors and death in animals. The avermectin group (anthelmintics, insecticides and acaricides) mediate the transmission of gamma-butyric acid (GABA), an inhibitory neurotransmitter, in mammals thus causing paralysis. Hepatotoxic effects with transient disturbances and jaundice have resulted from the use of oleandomycin. Transient alterations in heart rate/ rhythm have also been produced by several members of the family (notably tilmicosin). Heart muscle degeneration, characterized by small areas of cell death has also been reported in animals exposed to tilmicosin. Cross-resistance is often observed between the macrolide, lincosamide and streptogramin group of antibiotics.

'''Eye:'''
Although the material is not thought to be an irritant, direct contact with the eye may cause transient discomfort characterized by tearing or conjunctival redness (as with windburn). Slight abrasive damage may also result. The material may produce foreign body irritation in certain individuals.

'''Skin:'''
The material is not thought to produce adverse health effects or skin irritation following contact (as classified using animal models). Nevertheless, good hygiene practice requires that exposure be kept to a minimum and that suitable gloves be used in an occupational setting.

Open cuts, abraded or irritated skin should not be exposed to this material. Entry into the blood-stream through, for example, cuts, abrasions or lesions, may produce systemic injury with harmful effects. Examine the skin prior to the use of the material and ensure that any external damage is suitably protected.

'''Inhaled:'''
The material is not thought to produce either adverse health effects or irritation of the respiratory tract following inhalation (as classified using animal models). Nevertheless, adverse effects have been produced following exposure of animals by at least one other route and good hygiene practice requires that exposure be kept to a minimum and that suitable control measures be used in an occupational setting.

Persons with impaired respiratory function, airway diseases and conditions such as emphysema or chronic bronchitis, may incur further disability if excessive concentrations of particulate are inhaled.

==Engineering Controls==
*Enclosed local exhaust ventilation is required at points of dust, fume or vapor generation.
*Work should be performed in a fume hood or Biological Safety Cabinet.

==Work Practice Controls==
Decontamination should consist of surface cleaning with water and detergent followed by thorough rinsing. The use of detergent is recommended because there is no single accepted method of chemical deactivation for all agents involved. A plastic backed absorbent pad should be placed under the work area during the process. This should be changed at the end of each procedure or when a spill occurs.

==Personal Protective Equipment (PPE)==
Personal protective equipment includes a lab coat, eye protection and disposable gloves. Gloves should be changed frequently and should be pulled up over sleeves if possible to reduce the likelihood of any exposed skin. Wash hands and arms immediately after working with the substance. Particulate respirator protection should be assessed where incidental or accidental exposure is anticipated.

Note: Respirators are masks designed to protect the wearer from specific airborne hazards and are different from surgical masks, which protect the wearer only from splashes and are primarily intended to protect others from infectious aerosols exhaled by the wearer. Respirator use requires employee participation in the Respiratory Protection Program, which involves medical clearance and annual fit testing and training. Please be clear about use of surgical masks versus respirators. (Do NOT use the vague term “masks”.)

==Transportation and Storage==
*Keep containers tightly closed and store in a dry and ventilated area.
*Store away from strong oxidizing agents.
*Store according with information on product insert.
*Rapamycin is NOT REGULATED by DOT or IATA.

==Waste Disposal==
All hazardous chemical agent contaminated waste should be placed in a 5 gallon white pail. The container should be closed except when actively adding waste. The container must be located in the area where hazardous drugs are being used. All items contaminated by hazardous drugs, including gloves, syringes, vials needles, and solution containers should be disposed according to OSEH guidelines.

Sharps – place needles, syringes with needles attached and other breakable items into appropriately labeled sharps containers.

Empty stock vials, reagent bottles, etc. – triple rinse with copious amounts of water. Deface label with black magic marker or scraper. Place in a cardboard box for disposal.

Because most spent, unused and expired chemicals/materials are considered hazardous wastes, they must be properly disposed of. ''Do not dispose of chemical wastes by dumping them down a sink, flushing in a toilet or discarding in regular trash containers, unless authorized by OSEH Hazardous Materials Management (HMM).'''''Bold text''' Contact OSEH-HMM at (734) 763-4568 for waste containers, labels, manifests, waste collection and for any questions regarding proper waste disposal. Also refer to OSEH’s Hazardous Waste webpage for more information.

If animals are administered rapamycin in their food or water, both the food and water container must be labeled. Food or water that is not consumed by the animals must be collected, labeled and submitted for disposal as a hazardous material.

==Exposures/Unintended Contact==

'''If the employee is in need of emergency medical attention, call 911 immediately.
'''

'''SWALLOWED'''
*Seek medical advice.
*Give water to rinse out mouth.
*If swallowed do NOT induce vomiting, unless directed to do so by medical personnel.
*If vomiting occurs, lean patient forward or place on left side (head-down position, if possible) to maintain open airway and prevent aspiration.

'''EYE'''

If this product comes in contact with the eyes:
*Wash out immediately with fresh running water.
*Ensure complete irrigation of the eye by keeping eyelids apart and away from eye and moving the eyelids by occasionally lifting the upper and lower lids.
*If pain persists or recurs seek medical attention.
*Removal of contact lenses after an eye injury should only be undertaken by skilled personnel.

'''SKIN'''

If skin or hair contact occurs:
*Flush skin and hair with running water (and soap if available).
*Seek medical attention in event of irritation.

'''INHALED'''
*If inhaled remove from contaminated area.
*Consult a physician.

Contact OSEH for advice on symptoms of chemical exposure, or assistance in performing an exposure assessment.

Report all work related accidents, injuries, illnesses or exposures to WorkConnections within 24 hours by completing and submitting the Illness and Injury Report Form. Follow the directions on the WorkConnections website Forms Instructions to obtain proper medical treatment and follow-up.

Complete the OSEH Laboratory Incident and Near-Miss Report form.

==Treatment Facilities:==
*U-M Occupational Health Services -- Campus Employees
Mon-Fri 7:30 am - 4:30 pm
After hours - go to UM Hospital Emergency Dept. – Urgent Care Clinic
C380 Med Inn building
1500 East Medical Center Drive, Ann Arbor (734) 764-8021
*University Health Services -- University students (non-life threatening conditions)
Mon-Fri 8 am – 4:30 pm, Sat 9 am – 12 pm
Contact for current hours as they may vary
207 Fletcher Street, Ann Arbor (734) 764-8320
*UMHS Emergency Department -- after clinic hours or on weekends
1500 East Medical Center Drive, Ann Arbor, (734) 936-6666

Click here for additional accident and injury reporting information.

==Spill Procedure==
• When a spill occurs, '''personal safety should always come first'''''Italic text''.
• Alert and clear everyone in the immediate area where the spill occurred.

A minor (small) chemical spill is one that the laboratory staff is capable of handling safely without the assistance of safety and emergency personnel. A '''major/large chemical spill'''''Italic text'' requires active assistance from emergency personnel.

==Spill Response Steps:==

'''MINOR CHEMICAL SPILL'''
*Alert people in immediate area of spill.
*Open outside windows, if possible.
*Use proper personal protective equipment (PPE) as indicated above.
*Avoid breathing vapors from spill.
*Confine spill to as small an area as possible.
*Do not wash spill down the drain.
*Use appropriate spill kits/sorbents to absorb spill. Collect contaminated materials and residues and place in container. For powdered chemicals sweep carefully to avoid generation of dust or, if appropriate, use moist sorbent pads or wet the powder with a suitable solvent and then wipe with a dry cloth. Contact OSEH-HMM (734) 763-4568 for proper disposal.
*Clean spill area with water.

'''MAJOR CHEMICAL SPILL'''
*Attend to injured or contaminated persons and remove them from exposure.
*Alert people in the laboratory to evacuate.
*If spilled material is flammable, turn off ignition and heat sources. Don’t light Bunsen burners or turn on other switches.
*Call University of Michigan Division of Public Safety and Security (DPSS) at 911 immediately for assistance.
*Close doors to affected area.
*Post warnings to keep people from entering the area.
*Have person available that has knowledge of incident and laboratory to assist emergency personnel.

==Additional Spill Links:==
*www.oseh.umich.edu/pdf/chemspil.pdf
*http://www.oseh.umich.edu/emer-chemical.shtml.

'''Report all emergencies, suspicious activity, injuries, spills, and fires to the University of Michigan Division of Public Safety and Security (DPSS) by calling 911 or texting 377911. Register with the University of Michigan Emergency Alert System via Wolverine Access.
'''

==Training of Personnel==
All personnel are required to complete the General Laboratory Safety Training session (BLS025w or equivalent) via OSEH’s My LINC website. Furthermore, all personnel shall read and fully adhere to this SOP when handling rapamycin.

==Certification==
I have read and understand the above SOP. I agree to contact my Supervisor or Lab manager if I plan to modify this procedure. Sign by logging in and typing <nowiki>* ~~~~</nowiki> in the list below:

*[[User:Reddj|Reddj]] ([[User talk:Reddj|talk]]) 16:12, 13 October 2016 (UTC)
* [[User:Snyderds|Snyderds]] ([[User talk:Snyderds|talk]]) 19:37, 7 June 2017 (UTC)
* [[User:Elhabbal|Elhabbal]] ([[User talk:Elhabbal|talk]]) 21:53, 12 June 2017 (UTC)
*[[User:Mollyec|Mollyec]] ([[User talk:Mollyec|talk]]) 16:49, 6 June 2022 (UTC)
Prior Approval required – Is this procedure hazardous enough to warrant prior approval from the Laboratory Director? ☐ YES ☒ NO

Laboratory Director - Dave Bridges Revision Date - 2016-10-13

SOP - Cytotoxins and Antineoplastic Drugs

2022-06-06T16:48:02Z

Mollyec:

[[ Category: SOP ]]
[[ Category: Lab Safety]]

'''Principal Investigator (PI) Approval is Required Prior to Performing this Procedure'''
=Description=
This standard operating procedure outlines the handling and use of cytotoxic chemicals and antineoplastic drugs. Review this document and supply the information required in order to make it specific to your facility. In accordance with this document, laboratories should use appropriate controls, protective equipment, and disposal techniques when handling cytotoxic chemicals and antineoplastic drugs.

=Potential Hazards=
Although little research has been done on the long-term risk of occupational exposure to cytotoxic drugs, these drugs have been associated with human cancers at high (therapeutic) levels of exposure. These drugs have been shown to be carcinogens, mutagens and teratogens in many animal species. There is evidence that hazardous drugs may cause spontaneous abortions and increase the risk of congenital malformations. In addition, some of these drugs have been shown to cause acute effects in humans, such as localized skin necrosis (death of tissue), damage to normal skin after surface contact, dizziness, lightheadedness.

Antineoplastic drugs are substances used as a cancer chemotherapeutic agent which affect the reproductive capabilities including chromosomal damage (mutagens), effects on the fetus (teratogens), and are carcinogenic in animal models. Most hazardous drugs either bind directly to genetic material in the cell nucleus or affect cellular protein synthesis. Cytotoxic drugs may not distinguish between normal and cancerous cells. The growth and reproduction of the normal cells are often affected during treatment of cancerous cells.

=Engineering Controls=
All hazardous drug work will be conducted in a chemical fume hood or Class II Biological Safety Cabinet.

=Work Practice Controls=
Decontamination should consist of surface cleaning with water and detergent followed by thorough rinsing. The use of detergent is recommended because there is no single accepted method of chemical deactivation for all agents involved. A plastic backed absorbent pad should be placed under the work area during the process. This should be changed at the end of each process or when a spill occurs.

All locations within the laboratory where cytotoxic or antineoplastic chemicals are handled should be demarcated with designated area caution tape and/or posted with designated area caution signs. This includes all fume hoods and bench tops where the antineoplastic drugs are handled.

=Personal Protective Equipment=
Use doubled gloves with nitrile or neoprene gloves. Gloves should be changed frequently. Laboratory coat and safety glasses are required for preparation of material.
Laboratory SOP: Cytotoxins 1 Revision Date: 6/12/2014

=Waste Disposal=
All hazardous drug contaminated waste should be placed in a 5-gallon white pail. The container must be closed except when actively adding waste. The container must be located in the area where hazardous drugs are being used. All items contaminated by hazardous drugs, including gloves, syringes, vials, needles, and solution containers should be disposed according to Environment, Health and Safety (EHS) guidelines. Contact EHS at (810) 766-6763 for waste pick-up. Refer to UM-Flint Hazardous Waste Management Program and EHS webpage http://www.umflint.edu/ehs/environment- health-and-safety for more information.
Sharps – place needles, syringes with needles attached and other breakable items into appropriately labeled sharps containers.
Empty stock vials, reagent bottles, etc. – triple rinse with copious amounts of water. Deface label with black magic marker or scraper. Place in a cardboard box for disposal. Label the box as uncontaminated glass.

=Exposures/Unintended Contact=

For an actual chemical exposure/injury:
• Flush exposed eyes or skin with water for at least 15 minutes, then seek medical attention (see below).

• Note: In case of inhalation, symptoms may be delayed up to 24 hours. Seek medical attention.

• If swallowed, obtain urgent medical attention.

Contact EHS for advice on symptoms of chemical exposure, or assistance in performing an exposure assessment.
add here...
===TREATMENT FACILITIES:===

=== MAJOR INJURIES===
'''Genesys Hospital'''
One Genesys Parkway Grand Blanc, MI 48439 (810) 606-5710

'''Hurley Medical Center'''
One Hurley Plaza Flint, MI 48503 (810) 262-9000

'''McLaren Hospital Flint'''
401 South Ballenger Hwy Flint, MI 48532
(810) 342-2000
===MINOR INJURIES –During Business Hours===
'''Genesys Occupational Health Network'''
1460 Center Rd.
Burton, MI 48509
(810) 715-4620
Mon. to Fri. 7:30 am to 10 pm Sat. & Sun. Noon to 8 pm

'''McLaren Flint-Burton OCC Center'''
1459 S. Center Rd. Burton, MI 48509
(810) 496-0900
Mon. - Fri. 8 am to 8 pm Sat & Sun 10 am to 2 pm

===MINOR INJURIES –After Business Hours===
'''Downtown Flint'''
420 S. Saginaw St. Flint, MI 48502 (810) 762-1550

'''Genesys East'''
1096 S. Belsay Rd, Suite F Burton, MI 48509
(810) 743-3351

'''Genesys North'''
4154 W. Vienna Rd Clio, MI 48420 (810) 686-7397

'''Genesys South'''
8447 N. Holly Rd
Grand Blanc, MI 48439
(810) 603-0856
Mon. - Fri. 6 to 10pm / Sat. & Sun. 1-10pm

Click here for more information on the UM – Flint Emergency Preparedness and Response Plan.

=Spill Procedure=
• When a spill occurs, personal safety should always come first.

• Alert and clear everyone in the immediate area where the spill occurred.

A minor (small) chemical spill is one of a known chemical that the laboratory staff is capable of handling safely without the assistance of safety and emergency personnel, i.e., less than 1 Gallon or 3.5 Liters. A major/large chemical spill requires active assistance from emergency personnel.
'''Spill Response Steps:'''
===MINOR CHEMICAL SPILL===
• Alert people in immediate area of spill.

• If spilled material is flammable, turn off ignition and heat sources. Don’t light Bunsen burners or turn on other
switches.

• Open outside windows, if possible.

• Wear protective equipment, including safety goggles, gloves and long-sleeve lab coat.

• Avoid breathing vapors from spill.

• Confine spill to as small an area as possible.

• Do not wash spill down the drain.

•Use appropriate spill kits/sorbents to neutralize corrosives and/or absorb spill. Collect contaminated materials and residues and place in container. For powdered chemicals sweep carefully to avoid generation of dust or, if appropriate, use moist sorbent pads or wet the powder with a suitable solvent and then wipe with a dry cloth.

Contact EHS at (810) 766-6763 for proper disposal.

• Clean spill area with water.

===MAJOR CHEMICAL SPILL===
• Attend to injured or contaminated persons and remove them from exposure.

• Alert people in the laboratory to evacuate.

• If spilled material is flammable, turn off ignition and heat sources. Don’t light Bunsen burners or turn on other switches.

• Call UM-Flint DPS at 911 from any university telephone or (810) 762-3333 from any cell phone or non- university telephone immediately for assistance.

• Close doors to affected area.

• Post warnings to keep people from entering the area.

• Have person available that has knowledge of incident and laboratory to assist emergency personnel.

Additional Spill Links:

• www.oseh.umich.edu/pdf/chemspil.pdf

• http://www.oseh.umich.edu/emer-chemical.shtml.

Report all emergencies, suspicious activity, injuries, spills, and fires to the UM-Flint Department of Public Safety (DPS) at 911 from any university telephone or (810) 762-3333 from cell phone or non-university telephone. Register with the University of Michigan-Flint Emergency Alert System via Wolverine Access. Also, preprogram the UM-Flint DPS telephone number (810) 762-3333 into your cell phone for quick, easy use.

=Training of Personnel=
All personnel are required to complete Laboratory Safety Training. Documentation of the training is required. This training can be accomplished by completing the Comprehensive Laboratory Safety session (BLS009 or equivalent) via MyLINC, or UM-Flint EHS on-line training or other equivalent approved by EHS. Furthermore, all personnel shall read and fully adhere to this SOP when handling cytotoxins and antineoplastic drugs.

=Certification=
I have read and understand the above SOP. I agree to contact my Supervisor or Lab Manager if I plan to modify this procedure.

*[[User:Iharvey|Iharvey]] ([[User talk:Iharvey|talk]]) 21:12, 10 November 2016 (UTC)
*[[User:Elhabbal|Elhabbal]] ([[User talk:Elhabbal|talk]]) 22:02, 12 June 2017 (UTC)
*[[User:Reddj|Reddj]] ([[User talk:Reddj|talk]])
*[[User:Mollyec|Mollyec]] ([[User talk:Mollyec|talk]]) 16:48, 6 June 2022 (UTC)
Principal Investigator Revision Date
Laboratory SOP: Cytotoxins 4 Revision Date: 6/12/2014

SOP - BVT.2733

2022-06-06T16:47:02Z

Mollyec:

OSEH Recommendations/Findings for BVT.2733

Potential hazards of this material are unknown. This may be due to lack of information provided on the specific drug and/or it may be investigational/experimental/novel in nature. Therefore, limited or no information regarding potential toxicity was found. Thus, it is always prudent to handle as potentially hazardous agents. Caution should be exercised when handling and preparing solutions. The lab must use a biological safety cabinet during reconstitution and dilution of this material as well as use proper techniques to prevent contamination onto work surfaces. Personnel preparing this agent should wear double nitrile or latex gloves, safety glasses and a lab coat. All needles, syringes, vials, ampules and other materials which have come in contact with this agent should be segregated and disposed as hazardous waste. Unless evidence is provided to support a different course of action, it is EHS’s policy to house animals in the ULAM Animal Containment Housing facilities for a period of 3 days after each administration. After this time, the animals must be changed to a clean cage and can then be moved to standard animal housing facility. All waste materials, including bedding and carcasses must be collected and disposed through EHS HazMat while the animals are in Containment Housing. EHS HazMat (3‐4568) must be contacted prior to the commencement of this experiment to ensure proper waste containers are available for collection of this material. This policy is designed to reduce the potential exposure of animal care technicians and cage wash personnel to hazardous materials that may contaminate the dirty bedding. EHS can reevaluate housing requirements if the lab can provide information indicating that the material(s) are metabolized to a non‐hazardous substance and poses little risk to animal care personnel. Follow the link below for help on the preparation of chemical waste for collection: http://www.ehs.umich.edu/pdf/Chemical_Waste_Poster.pdf

[[ Category: SOP ]]
[[ Category: Lab Safety ]]

* [[User:Elhabbal|Elhabbal]] ([[User talk:Elhabbal|talk]]) 21:48, 12 June 2017 (UTC)
*[[User:Mollyec|Mollyec]] ([[User talk:Mollyec|talk]]) 16:47, 6 June 2022 (UTC)

SOP - Bunsen Burners

2022-06-06T16:45:50Z

Mollyec:

[[ Category: SOP ]]
[[ Category: Lab Safety ]]

==Description==
This standard operating procedure outlines the handling and use of Bunsen burners. Review this document and supply the information required in order to make it specific to your laboratory. In accordance with this document, laboratories should use appropriate controls and personal protective equipment when using Bunsen burners.

Bunsen burners produce a single open flame by burning a continuous stream of flammable gas used for heating, sterilization, and combustion.

==Potential Hazards==
Bunsen burners present burn and fire hazards due to the high-temperature open flame that is produced.

==Engineering Controls==
Biological safety cabinets (BSCs) and disposable sterile items obviate the need for open flames when aseptic conditions are needed. The use of a Bunsen burner in a BSC creates thermodynamic instabilities that interfere with the function of the unit, reducing its ability to provide the sterile field. If an open flame absolutely must be used in a BSC, recommended alternatives such as electrical incinerators or touch-plate microburners are available.

==Work Practice Controls==
Bunsen burner safety – best practice:
* PLACE the Bunsen burner away from any overhead shelving, equipment, or light fixtures.
* REMOVE all papers, notebooks, combustible materials and excess chemicals from the area.
* TIE-BACK any long hair, dangling jewelry, or loose clothing.
* INSPECT hose for cracks, holes, pinched points, or any other defect and ensure that the hose fits securely on the gas valve and the Bunsen burner.
* REPLACE all hoses found to have a defect before using.
* NOTIFY others in the laboratory that burner will be in use.
* UTILIZE a sparker/lighter with extended nozzle to ignite the Bunsen burner. Never use a match to ignite burner.
* HAVE the sparker/lighter available before turning on gas.
* ADJUST the flame by turning the collar to regulate air flow and produce an appropriate flame for the experiment (typically a medium blue flame).
* DO NOT leave open flames unattended and never leave laboratory while burner is on.
* SHUT-OFF gas when its use is complete.
* ALLOW the burner to cool before handling.
* ENSURE that the main gas valve is off before leaving the laboratory.

Additional Precautions:
* Do not use a Bunsen burner in biological safety cabinet.
* For small fires, attempt to extinguish fire if you been trained in fire extinguisher use.
* In case of a large fire activate the fire alarm, evacuate the building and alert authorities.

==Personal Protective Equipment (PPE)==
Wear standard laboratory attire including safety glasses and avoid wearing synthetic clothing.

==Exposures/Unintended Contact==

If the employee is in need of emergency medical attention, call 911 immediately.

Contact OSEH for advice on symptoms of chemical exposure, or assistance in performing an exposure assessment.

Report all work related accidents, injuries, illnesses or exposures to WorkConnections within 24 hours by completing and submitting the Illness and Injury Report Form. Follow the directions on the WorkConnections website Forms Instructions to obtain proper medical treatment and follow-up.

Complete the OSEH Laboratory Incident and Near-Miss Report form.

TREATMENT FACILITIES:
U-M Occupational Health Services -- Campus Employees
Mon-Fri 7:30 am - 4:30 pm
After hours - go to UM Hospital Emergency Dept. – Urgent Care Clinic
C380 Med Inn building
1500 East Medical Center Drive, Ann Arbor (734) 764-8021
University Health Services -- University students (non-life threatening conditions)
Mon-Fri 8 am – 4:30 pm, Sat 9 am – 12 pm
Contact for current hours as they may vary
207 Fletcher Street, Ann Arbor (734) 764-8320
UMHS Emergency Department -- after clinic hours or on weekends
1500 East Medical Center Drive, Ann Arbor, (734) 936-6666
Click here for additional accident and injury reporting information.

==Release/Leak Procedure==
* When a gas leak occurs, personal safety should always come first.
* Alert and clear everyone in the immediate area where the gas leak occurred.
* Open outside windows, if possible & safe to do so.
* Avoid breathing gas.
* Contact OSEH-HMM (734) 763-4568 for release assistance.

Report all emergencies, suspicious activity, injuries, spills, and fires to the University of Michigan Division of Public Safety and Security (DPSS) by calling 911 or texting 377911. Register with the University of Michigan Emergency Alert System via Wolverine Access.

==Training of Personnel==
All personnel are required to complete the General Laboratory Safety Training session (BLS025w or equivalent) via OSEH’s My LINC website. Furthermore, all personnel shall read and fully adhere to this SOP when working with Bunsen burners.

Certification
I have read and understand the above SOP. I agree to contact my Supervisor or Lab Manager if I plan to modify this procedure. Sign by logging in and typing <nowiki>* ~~~~</nowiki> in the list below:
* [[User:Ccousz|Ccousz]] ([[User talk:Ccousz|talk]]) 16:58, 12 October 2016 (UTC)
* [[User:Davebridges|Davebridges]] ([[User talk:Davebridges|talk]]) 14:50, 8 November 2016 (UTC)
*[[User:Iharvey|Iharvey]] ([[User talk:Iharvey|talk]]) 21:17, 10 November 2016 (UTC)
* [[User:Elhabbal|Elhabbal]] ([[User talk:Elhabbal|talk]]) 21:46, 12 June 2017 (UTC)
*[[User:Reddj|Reddj]] ([[User talk:Reddj|talk]])
*[[User:Mollyec|Mollyec]] ([[User talk:Mollyec|talk]]) 16:45, 6 June 2022 (UTC)
Prior Approval required – Is this procedure hazardous enough to warrant prior approval from the Laboratory Director? ☐ YES X NO

Laboratory Director - Dave Bridges Revision Date - 2016-10-12

QPCR

2022-04-05T19:18:35Z

Mollyec:

__NOTOC__
==Real Time qPCR==
===Materials===
*cDNA: see [[First_Strand_cDNA_Synthesis_(AB_Kit)|First Strand cDNA Synthesis (AB Kit)]] for details
*SYBR Green PCR Master Mix Applied Biosystems (ThermoFisher Catalog # 4367659; [https://www.thermofisher.com/order/catalog/product/4367659 Vendor Link])
*384 well qPCR plate (ThermoFisher Catalog # 4309849) and covers (Catalog # 4360954)
*Primers (Dilute to 0.4 uM mixture of fwd and rev. From 100 uM stocks- the 100uM stock is prepared by adding 227 uL of distilled water to 22.7nmol of a gene as an example- this is 4uL Forward Primer, 4 uL Reverse Primer and 992 uL Water). This primer mix can be stored at -20 in a Working Primers box. Design primers according to [[Primer Design for qPCR]]

===Plate Preparation===
#Book 2h on qPCR machine by signing up on the sheet in room 7013 and on the [https://calendar.google.com/calendar/embed?src=umich.edu_jkmcg58uidmngdh0sutt1d36og%40group.calendar.google.com&ctz=America%2FDetroit google calendar]
#Prepare cDNA and dilute in water in a 96 well plate. Typically a 20x dilution of cDNA leaves enough to be detected.
#Get optically clear 384 well plate and keep on paper towel. Do not touch bottom of plate.
#Sketch out the plate in your notes. Typically rows are different primers while columns are different cDNA's
#Calculate how many samples x how many replicates/per sample (start with 3 or 4 until you are consistent enough technically to decrease). This will be the number of wells need for each primer.
#Prepare a Primer/SYBR Green mixture for each primer. For each well you will need 5 uL SYBR green + 2.5 uL Primer working stock, so if you have calculated you need 10 wells per primer that is 50 uL SYBR Green + 25 uL Primers. Make up 10-20% more than you need.
#Using the repeater multichannel pipette put on 2 or 3 tips (depending on your plate arrangement) and set to aspirate however many samples you have and dispense 7.5 uL per well.
#Dispense 7.5 uL of Primer/SYBR mixture into each well, dispensing at the bottom of the well.
#Using the ClipTip multichannel add 2.5 uL of cDNA to each applicable well. You don't need to change tips between wells.
#Once the plate is completed, carefully put an optically clear cover on it using the plastic square to ensure the edges are sealed, being very careful not to leave fingerprints on the seal.
#You can prepare the plate ~3h beforehand, keeping it at 4C until the machine is ready.
#Immediately before the run spin the plate briefly (2 mins at 4000 RPM) in the swinging bucket centrifuge.

===Run Protocol===
* Set up run using [[Set up qPCR Run on Thermo Cloud and QuantStudio|Thermo Cloud/QuantStudio]] or [[Set up qPCR Run on Roche LightCycler|Roche LightCycler]]
Briefly, log into thermo cloud
*Select "Design and Analysis New, qPCR" from the home screen
*Select "set up plate"
*Select "comparative CT-SYBR"
*Correct volume to read, "10.0uL" instead of "20.0uL"
*Select "Plate set up"
*Navigate to the drop down menu marked "passive reference" and select "none."
*Then import [https://docs.google.com/spreadsheets/d/e/2PACX-1vQpOEYWkqKe6tELqIerA7fcwe2LoEQy_8ANGuhOppmGXbD0sBGvKXHnR3z7aYnfEF7--r4FbOd6yCYM/pub?output=csv plate setup] with linked CSV (MUST be a csv file).
*Save to the cloud and upload on the machine when you insert the plate.

==Calculations==
see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC55695 for considerations on calculations

[[ Category: Molecular Biology ]]
[[ Category: RNA ]]
[[ Category: qPCR ]]
[[ Category: Transcription ]]
[[ Category: Expression ]]

QPCR

2022-04-05T19:15:40Z

Mollyec:

__NOTOC__
==Real Time qPCR==
===Materials===
*cDNA: see [[First_Strand_cDNA_Synthesis_(AB_Kit)|First Strand cDNA Synthesis (AB Kit)]] for details
*SYBR Green PCR Master Mix Applied Biosystems (ThermoFisher Catalog # 4367659; [https://www.thermofisher.com/order/catalog/product/4367659 Vendor Link])
*384 well qPCR plate (ThermoFisher Catalog # 4309849) and covers (Catalog # 4360954)
*Primers (Dilute to 0.4 uM mixture of fwd and rev. From 100 uM stocks- the 100uM stock is prepared by adding 227 uL of distilled water to 22.7nmol of a gene as an example- this is 4uL Forward Primer, 4 uL Reverse Primer and 992 uL Water). This primer mix can be stored at -20 in a Working Primers box. Design primers according to [[Primer Design for qPCR]]

===Plate Preparation===
#Book 2h on qPCR machine by signing up on the sheet in room 7013 and on the [https://calendar.google.com/calendar/embed?src=umich.edu_jkmcg58uidmngdh0sutt1d36og%40group.calendar.google.com&ctz=America%2FDetroit google calendar]
#Prepare cDNA and dilute in water in a 96 well plate. Typically a 20x dilution of cDNA leaves enough to be detected.
#Get optically clear 384 well plate and keep on paper towel. Do not touch bottom of plate.
#Sketch out the plate in your notes. Typically rows are different primers while columns are different cDNA's
#Calculate how many samples x how many replicates/per sample (start with 3 or 4 until you are consistent enough technically to decrease). This will be the number of wells need for each primer.
#Prepare a Primer/SYBR Green mixture for each primer. For each well you will need 5 uL SYBR green + 2.5 uL Primer working stock, so if you have calculated you need 10 wells per primer that is 50 uL SYBR Green + 25 uL Primers. Make up 10-20% more than you need.
#Using the repeater multichannel pipette put on 2 or 3 tips (depending on your plate arrangement) and set to aspirate however many samples you have and dispense 7.5 uL per well.
#Dispense 7.5 uL of Primer/SYBR mixture into each well, dispensing at the bottom of the well.
#Using the ClipTip multichannel add 2.5 uL of cDNA to each applicable well. You don't need to change tips between wells.
#Once the plate is completed, carefully put an optically clear cover on it using the plastic square to ensure the edges are sealed, being very careful not to leave fingerprints on the seal.
#You can prepare the plate ~3h beforehand, keeping it at 4C until the machine is ready.
#Immediately before the run spin the plate briefly (2 mins at 4000 RPM) in the swinging bucket centrifuge.

===Run Protocol===
* Set up run using [[Set up qPCR Run on Thermo Cloud and QuantStudio|Thermo Cloud/QuantStudio]] or [[Set up qPCR Run on Roche LightCycler|Roche LightCycler]]
Briefly, log into thermo cloud
*Select "Design and Analysis New, qPCR" from the home screen
*Select "set up plate"
*Select "comparative CT-SYBR"
*Correct volume to read, "10.0uL" instead of "20.0uL"
*Select "Plate set up"
*Navigate to the drop down menu marked "passive reference" and select "none."
*Then import plate setup with linked CSV (MUST be a csv file).
*Save to the cloud and upload on the machine when you insert the plate.

==Calculations==
see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC55695 for considerations on calculations

[[ Category: Molecular Biology ]]
[[ Category: RNA ]]
[[ Category: qPCR ]]
[[ Category: Transcription ]]
[[ Category: Expression ]]

Milk Creamatocrit

2022-03-18T20:29:59Z

Mollyec:

==Mouse Milk Creamatocrit (milk fat %)==
This method is done to assess the total fat content of mouse milk. It does not differentiate between type of fat present. Additional information on mouse milk composition can be ascertained through triglyceride or cholesterol assay, or through lipidomics.
===Materials===
*Milk samples (from -80)
*Ice bucket
*CritSpin Micro-Hematocrit Spinner (from the Pennathur lab in Brehm tower)
*1XPBS + EDTA (50mL 1X PBS + 100uL 0.5M EDTA)
*Untreated glass hematocrit tubes
*Fractional dial calipers
*Critoseal
*Kimwipes
*P10 pipette with tips

===Preparing the samples===
#Thaw whole milk on ice. To homogenize thawed milk, pipette up and down for five seconds. DO NOT shake!
#Dilute the milk in aa 1:3 solution with the 1XPBS + EDTA. Make enough for 4 capillary tubes
#Take capillary tube and draw in enough milk to fill ~3/4 of the tube (stop before the blue line)
#Hold horizontally and seal the other end of the tube with critoseal twice. Be sure to continue to hold the tube horizontally, or the milk will be ejected.
#Wipe excess milk from the tube with the kimwipe
#Each sample should be run in duplicate (you may need to run additional tubes if the variance between the first 2 sample results is >=25%)
===Running samples===
* Place sealed and filled capillaries into the critspin micro-hematocrit spinner. The sealed end should point toward the outer edge of the centrifuge.
* Record the position of each tube/sample before running..
* Screw on the centrifuge cover so that it is secure, but not too difficult to loosen later.
* Spin for 120 seconds, eight separate times. Between spins, the centrifuge will come to a complete stop on its own.
* After the 8th spin, immediately read the creamatocrit using the calipers
===Reading the samples===
* Measure each of the 3 layers to the nearest 0.01 inch with the calipers. Record the number in the corresponding columns of [https://docs.google.com/spreadsheets/d/e/2PACX-1vTm1Zo5Cf9RTuw_uRwum8h_dOBb0mjc5HYcOHR1nBPHKbSzxlFqwYfJs0NMdChFHrczE_8sKCU3K9lp/pub?output=csv the spreadsheet](the aqueous layer(Aqueous.inches.1), the fat layer(Fat.inches), and the small aqueous layer on top of the fat layer(Aqueous.inches.2)).
* Calculate the milk fat percentage using the following R code:

```{r data-manipulation}
mutated.milk.data<-milk.data%>%
group_by(replicate, MouseID, Genotype)%>%
mutate(fat = Fat.inches*25.4,
water = (Aqueous.inches.1 + Aqueous.inches.2)*25.4)%>%
mutate(water.corrected = water/4)%>%
mutate(total.volume = water.corrected+fat)%>%
mutate(fat.percent = (fat/total.volume)*100)
```

Milk Creamatocrit

2022-03-17T15:44:23Z

Mollyec: /* Reading the samples */

==Mouse Milk Creamatocrit (milk fat %)==
===Materials===
*Milk samples (from -80)
*Ice bucket
*CritSpin Micro-Hematocrit Spinner (from the Pennathur lab in Brehm tower)
*1XPBS + EDTA (50mL 1X PBS + 100uL 0.5M EDTA)
*Untreated glass hematocrit tubes
*Fractional dial calipers
*Critoseal
*Kimwipes
*P10 pipette with tips

===Preparing the samples===
#Thaw whole milk on ice. To homogenize thawed milk, pipette up and down for five seconds. DO NOT shake!
#Dilute the milk in aa 1:3 solution with the 1XPBS + EDTA. Make enough for 4 capillary tubes
#Take capillary tube and draw in enough milk to fill ~3/4 of the tube (stop before the blue line)
#Hold horizontally and seal the other end of the tube with critoseal twice. Be sure to continue to hold the tube horizontally, or the milk will be ejected.
#Wipe excess milk from the tube with the kimwipe
#Each sample should be run in duplicate (you may need to run additional tubes if the variance between the first 2 sample results is >=25%)
===Running samples===
* Place sealed and filled capillaries into the critspin micro-hematocrit spinner. The sealed end should point toward the outer edge of the centrifuge.
* Record the position of each tube/sample before running..
* Screw on the centrifuge cover so that it is secure, but not too difficult to loosen later.
* Spin for 120 seconds, eight separate times. Between spins, the centrifuge will come to a complete stop on its own.
* After the 8th spin, immediately read the creamatocrit using the calipers
===Reading the samples===
* Measure to the nearest 0.01 inch the aqueous layer, the fat layer, and the small aqueous layer on top of the fat layer.
* Record results in a csv file.
* Calculate the milk fat percentage using the following R code:

```{r data-manipulation}
mutated.milk.data<-milk.data%>%
group_by(replicate, MouseID, Genotype)%>%
mutate(fat = Fat.inches*25.4,
water = (Aqueous.inches.1 + Aqueous.inches.2)*25.4)%>%
mutate(water.corrected = water/4)%>%
mutate(total.volume = water.corrected+fat)%>%
mutate(fat.percent = (fat/total.volume)*100)
```

Milk Creamatocrit

2022-03-17T15:43:00Z

Mollyec: added more to materials

==Mouse Milk Creamatocrit (milk fat %)==
===Materials===
*Milk samples (from -80)
*Ice bucket
*CritSpin Micro-Hematocrit Spinner (from the Pennathur lab in Brehm tower)
*1XPBS + EDTA (50mL 1X PBS + 100uL 0.5M EDTA)
*Untreated glass hematocrit tubes
*Fractional dial calipers
*Critoseal
*Kimwipes
*P10 pipette with tips

===Preparing the samples===
#Thaw whole milk on ice. To homogenize thawed milk, pipette up and down for five seconds. DO NOT shake!
#Dilute the milk in aa 1:3 solution with the 1XPBS + EDTA. Make enough for 4 capillary tubes
#Take capillary tube and draw in enough milk to fill ~3/4 of the tube (stop before the blue line)
#Hold horizontally and seal the other end of the tube with critoseal twice. Be sure to continue to hold the tube horizontally, or the milk will be ejected.
#Wipe excess milk from the tube with the kimwipe
#Each sample should be run in duplicate (you may need to run additional tubes if the variance between the first 2 sample results is >=25%)
===Running samples===
* Place sealed and filled capillaries into the critspin micro-hematocrit spinner. The sealed end should point toward the outer edge of the centrifuge.
* Record the position of each tube/sample before running..
* Screw on the centrifuge cover so that it is secure, but not too difficult to loosen later.
* Spin for 120 seconds, eight separate times. Between spins, the centrifuge will come to a complete stop on its own.
* After the 8th spin, immediately read the creamatocrit using the calipers
===Reading the samples===
* Measure to the nearest 0.01 inch the aqueous layer, the fat layer, and the small aqueous layer on top of the fat layer.
* Record results in a csv file.
* Calculate the milk fat percentage using the following R code:

mutated.milk.data<-milk.data%>%
group_by(replicate, MouseID, Genotype)%>%
mutate(fat = Fat.inches*25.4,
water = (Aqueous.inches.1 + Aqueous.inches.2)*25.4)%>%
mutate(water.corrected = water/4)%>%
mutate(total.volume = water.corrected+fat)%>%
mutate(fat.percent = (fat/total.volume)*100)

Milk Creamatocrit

2022-03-15T13:57:18Z

Mollyec: generated protocol from Brigid's creamatocrit SOP

==Mouse Milk Creamatocrit (milk fat %)==
===Materials===
*CritSpin Micro-Hematocrit Spinner (from the Pennathur lab in Brehm tower)
*1XPBS + EDTA (50mL 1X PBS + 100uL 0.5M EDTA)
*untreated glass hematocrit tubes
*Fractional dial calipers
*Critoseal
*Kimwipes
*P10 pipette with tips

===Preparing the samples===
#Thaw whole milk on ice. To homogenize thawed milk, pipette up and down for five seconds. DO NOT shake!
#Dilute the milk in aa 1:3 solution with the 1XPBS + EDTA. Make enough for 4 capillary tubes
#Take capillary tube and draw in enough milk to fill ~3/4 of the tube (stop before the blue line)
#Hold horizontally and seal the other end of the tube with critoseal twice. Be sure to continue to hold the tube horizontally, or the milk will be ejected.
#Wipe excess milk from the tube with the kimwipe
#Each sample should be run in duplicate (you may need to run additional tubes if the variance between the first 2 sample results is >=25%)
===Running samples===
* Place sealed and filled capillaries into the critspin micro-hematocrit spinner. The sealed end should point toward the outer edge of the centrifuge.
* Record the position of each tube/sample before running..
* Screw on the centrifuge cover so that it is secure, but not too difficult to loosen later.
* Spin for 120 seconds, eight separate times. Between spins, the centrifuge will come to a complete stop on its own.
* After the 8th spin, immediately read the creamatocrit using the calipers
===Reading the samples===
* Measure to the nearest 0.01 inch the aqueous layer, the fat layer, and the small aqueous layer on top of the fat layer.
* Record results in a csv file.
* Calculate the milk fat percentage using the following R code:

mutated.milk.data<-milk.data%>%
group_by(replicate, MouseID, Genotype)%>%
mutate(fat = Fat.inches*25.4,
water = (Aqueous.inches.1 + Aqueous.inches.2)*25.4)%>%
mutate(water.corrected = water/4)%>%
mutate(total.volume = water.corrected+fat)%>%
mutate(fat.percent = (fat/total.volume)*100)

Rate of drop analysis

2020-07-28T13:48:05Z

Mollyec: /* Sample R Code */

==Purpose==
To assess the initial response to insulin administration, you can calculate the slope of the line for the early timepoints in your insulin tolerance test. To do this, complete the following steps:

==Methods==
1. First, filter your ITT data to contain only the observations from where animals are in the initial drop of the experiment. This is usually 30-45 minutes, but you should assess this for each ITT yourself.

2. You then need to construct a linear model for your data. It should include 1 covariate for every experimental variable (eg, time, genotype, sex, diet, injection). You should also include an interaction variable for time and the variable of interest. This will be the crucial value that you interpret different slopes of the initial drop from.

3. After creating the model, you will need to assess the significance of that model. Do this using the anova command in R. This will give you the covariate p values.

4. Next, to find the beta estimates, you will need to run the coefficient command in R. This will tell you the actual changes in glucose (mg/dL) for each covariate. The beta coefficient of interest is the one corresponding to the interaction variable you coded in step 2.

==Sample R Code==
new.data.set <-original.data.set%>%
filter(time=="0"| time == "15" | time == "30"| time =="45")

drop.data.lm <- lm (formula = ( glucose ~ time + treatment + time:treatment, data = new.data.set)

anova(drop.data.lm)

coefficients(drop.data.lm)

Rate of drop analysis

2020-04-01T18:19:07Z

Mollyec: /* Sample R Code */

==Purpose==
To assess the initial response to insulin administration, you can calculate the slope of the line for the early timepoints in your insulin tolerance test. To do this, complete the following steps:

==Methods==
1. First, filter your ITT data to contain only the observations from where animals are in the initial drop of the experiment. This is usually 30-45 minutes, but you should assess this for each ITT yourself.

2. You then need to construct a linear model for your data. It should include 1 covariate for every experimental variable (eg, time, genotype, sex, diet, injection). You should also include an interaction variable for time and the variable of interest. This will be the crucial value that you interpret different slopes of the initial drop from.

3. After creating the model, you will need to assess the significance of that model. Do this using the anova command in R. This will give you the covariate p values.

4. Next, to find the beta estimates, you will need to run the coefficient command in R. This will tell you the actual changes in glucose (mg/dL) for each covariate. The beta coefficient of interest is the one corresponding to the interaction variable you coded in step 2.

==Sample R Code==
new.data.set <-original.data.set%>%
filter(time=="0"| time == "30"| time =="45")

drop.data.lm <- lm (formula = ( glucose ~ time + treatment + time:treatment, data = new.data.set)

anova(drop.data.lm)

coefficients(drop.data.lm)

Rate of drop analysis

2020-04-01T18:18:50Z

Mollyec:

==Purpose==
To assess the initial response to insulin administration, you can calculate the slope of the line for the early timepoints in your insulin tolerance test. To do this, complete the following steps:

==Methods==
1. First, filter your ITT data to contain only the observations from where animals are in the initial drop of the experiment. This is usually 30-45 minutes, but you should assess this for each ITT yourself.

2. You then need to construct a linear model for your data. It should include 1 covariate for every experimental variable (eg, time, genotype, sex, diet, injection). You should also include an interaction variable for time and the variable of interest. This will be the crucial value that you interpret different slopes of the initial drop from.

3. After creating the model, you will need to assess the significance of that model. Do this using the anova command in R. This will give you the covariate p values.

4. Next, to find the beta estimates, you will need to run the coefficient command in R. This will tell you the actual changes in glucose (mg/dL) for each covariate. The beta coefficient of interest is the one corresponding to the interaction variable you coded in step 2.

==Sample R Code==
new.data.set <-original.data.set%>%
filter(time=="0"| time == "30"| time =="45")

drop.data.lm <- lm (formula = ( glucose ~ time + treatment + time:treatment, data = new.data.set, )

anova(drop.data.lm)

coefficients(drop.data.lm)

Rate of drop analysis

2020-04-01T18:07:40Z

Mollyec: Created page with "==Purpose== To assess the initial response to insulin administration, you can calculate the slope of the line for the early timepoints in your insulin tolerance test. To do th..."

==Purpose==
To assess the initial response to insulin administration, you can calculate the slope of the line for the early timepoints in your insulin tolerance test. To do this, complete the following steps:

==Methods==
1. First, filter your ITT data to contain only the observations from where animals are in the initial drop of the experiment. This is usually 30-45 minutes, but you should assess this for each ITT yourself.

2. You then need to construct a linear model for your data. It should include 1 covariate for every experimental variable (eg, time, genotype, sex, diet, injection). You should also include an interaction variable for time and the variable of interest. This will be the crucial value that you interpret different slopes of the initial drop from.

3. After creating the model, you will need to assess the significance of that model. Do this using the anova command in R. This will give you the covariate p values.

4. Next, to find the beta estimates, you will need to run the coefficient command in R. This will tell you the actual changes in glucose (mg/dL) for each covariate. The beta coefficient of interest is the one corresponding to the interaction variable you coded in step 2.

==Sample R Code==
new.data.set <-original.data.set%>%
filter(time=="0"| time == "30"| time =="45")

drop.data.lm <- lm (data = new.data.set, formula = ( glucose ~ time + treatment + time:treatment)

anova(drop.data.lm)

coefficients(drop.data.lm)

Spill Response Procedures for Infectious agents and Recombinant DNA

2019-09-12T16:14:32Z

Mollyec: /* Certification */

Spills and accidents should be immediately reported to the Principal Investigator and the Department of Occupational Safety and Environmental Health (OSEH) (647-1143).

Large Spills Outside of the Biosafety Cabinet
OSEH will respond to large spills (greater than 200 ml) of BSL-2 (or above) containment level material. Contact OSEH (647-1143) for help with large spill cleanup.
Small Spills Outside of the Biosafety Cabinet
# Don double gloves and eye protection if not already wearing them.
# Cover spilled material with an absorbent paper towel or Kimwipe. Once the absorbent material is in place over the spill, wet the material with a 10% solution of bleach (1:10 dilution of Clorox containing sodium hypochlorite) or other appropriate disinfectant.
# Let stand 15-20 minutes, wipe up and wash surface with appropriate disinfectant.
# Wipe down all equipment and surfaces which may have been splashed.
Spill in a Biosafety Cabinet
Note: Leave the cabinet turned on.
# Don double gloves, a lab coat, and eye protection if not already wearing them.
# Cover spilled material with an absorbent paper towel or Kimwipe. Once the absorbent material is in place, wet material with 10% solution of bleach (1:10 dilution of Clorox containing sodium hypochlorite) or other appropriate disinfectant. Let stand 15-20 minutes, wipe up and wash surface with appropriate disinfectant.
# If personnel are contaminated, remove potentially contaminated garments at the BSC and decontaminate garments by saturation with 70% ethanol or place in autoclave bag for autoclaving. Wash hands and other potentially exposed skin surfaces thoroughly with soap and water. Don fresh PPE, return to worksite, and spray walls, liners, and equipment with an appropriate disinfectant.
# Spray or wipe container walls, other work surfaces and equipment with the appropriate disinfectant
# If necessary, flood the work surface, drain pan and catch basin below the work surface with disinfectant. Allow at least 15-20 minutes contact time.
# Soak up the disinfectant and drain the catch basin into a container. Lift the front exhaust grille and tray and wipe all surfaces. Ensure that no foreign materials are blown into the area below the grille.
# If a 10% bleach solution is used on metal surfaces, rinse with water or 70% ethanol after decontamination is complete.
# If the spill overflows into the interior of the cabinet, more extensive decontamination of the cabinet may be necessary. Contact OSEH (763-6973) for decontamination of the cabinet.
Spill in the Laboratory
# If an infectious aerosol may exist outside of a biosafety cabinet, leave the room immediately, lock the door, post a warning sign and inform your supervisor. If clothing is contaminated, remove and turn the exposed side of fabric in on itself and place in autoclave bag or biohazard container. Wait at least 30 minutes before reentering the lab to allow dissipation of aerosol created by the spill. During this time, review clean-up procedures, assemble decontamination materials, PPE and contact OSEH (647-1143).
# Don fresh gloves, a lab coat or tyvek suit, and eye protection.
# Carefully lay disinfectant-soaked towels over the spill and pour more around the spill. Use more concentrated disinfectant if the volume of material will significantly dilute the disinfectant.
# Allow 15-20 minutes contact time.
# Use forceps to place sharp objects into a sharps container. Using a dustpan and dustbroom, tongs, etc., transfer all contaminated materials (paper towels, gloves, labware, etc.) biohazard waste containers and contact OSEH HazMat (763-4568) for removal.
# Wipe surrounding surfaces with disinfectant to cover all splash areas. Wipe flat surfaces to remove any material that may have splashed out and settled on those surfaces.
# Place all contaminated materials, including protective clothing, into an autoclave bag or biohazard waste container.
# Wash hands with soap and warm water.
# If a personnel exposure occurred, complete an Illness or Injury Report form (https://www.workconnections.umich.edu/illnessorinjury.php) summit to Work~Connections and report incident to OSEH (763-6973). See additional procedures noted below.
Skin, Mucous Membrane, or Injury Exposure
# If there is an agent-specific protocol for exposures, follow that (e.g., HIV, Herpes B).
# In the case of skin contact or injury with a contaminated instrument:
## Thoroughly wash area with soap and water. Do not squeeze the wound to induce bleeding.
## Avoid use of abrasive chemical soaps or disinfectant washes as they can cause skin abrasions and a possible additional route of entry for the agent.
## For mucous membranes (e.g., eyes, mouth), flush for a minimum of 15 minutes.
# Notify UM-Occupational Health Services (OHS) for evaluation of exposure following:
## Contact with mucous membranes;
## Contact with non-intact skin;
## Percutaneous exposure; or
## Any type of exposure that involves concentrated virus.

==Certification==
I have read and understand the above SOP. I agree to contact my Supervisor or Lab manager if I plan to modify this procedure. Sign by logging in and typing <nowiki>* ~~~~</nowiki> in the list below:

* [[User:Davebridges|Davebridges]] ([[User talk:Davebridges|talk]]) 14:44, 13 September 2016 (UTC)
*[[User:Iharvey|Iharvey]] ([[User talk:Iharvey|talk]]) 21:28, 10 November 2016 (UTC)
* [[User:Snyderds|Snyderds]] ([[User talk:Snyderds|talk]]) 19:58, 7 June 2017 (UTC)
* [[User:Elhabbal|Elhabbal]] ([[User talk:Elhabbal|talk]]) 22:21, 12 June 2017 (UTC)
*[[User:Reddj|Reddj]] ([[User talk:Reddj|talk]]) 17:26, 15 June 2017 (UTC)
*[[User:Mollyec|Mollyec]] ([[User talk:Mollyec|talk]]) 21:43, 12 September 2019 (UTC)
Prior Approval required – Is this procedure hazardous enough to warrant prior approval from the Laboratory Director? ☐ YES X NO

Laboratory Director - Dave Bridges
Revision Date - 2016-09-13

[[ Category:SOP ]]

SOP - Phenol

2019-09-12T16:13:50Z

Mollyec: /* Spill Procedure */

[[Category: SOP]]
[[Category: Lab Safety]]
'''Laboratory Director (LD) Approval is Required Prior to Performing this Procedure'''

=Description=
This standard operating procedure (SOP) outlines the handling and use of phenol. Review this document and supply the information required in order to make it specific to your laboratory. In accordance with this document, laboratories should use appropriate controls, personal protective equipment, and disposal techniques when handling phenol.

Phenol is used as a reagent in chemical analysis. It causes burns and risk of serious damage to eyes. Phenol is toxic by inhalation, in contact with skin and if swallowed.

=Potential Hazards=
• Phenol is very hazardous in case of skin contact – it is corrosive, toxic, and can cause irritation. Initially, it can cause numbness or slight tingling, so employees may not be immediately aware of contact. If absorbed through the skin it can cause muscle weakness, tremors, loss of coordination, shock, sudden collapse, coma, convulsions, organ damage, and death. It may cause severe eye injury (including blindness) if it contacts the eyes, and is extremely toxic (fatal) by ingestion.

• Inhalation exposure is less likely – it does not evaporate easily at room temperature, but can be inhaled if heated and/or misted, or in the case of a large spill. If inhaled, phenol can cause upper respiratory irritation, lung damage, and CNS impairment.

• For more safety information, refer to Prudent Practice’s Laboratory Chemical Safety Summary for Phenol.

Occupational Exposure Limits (OELs):
• MIOSHA PEL and ACGIH TLV: 5 ppm, 8-hour TWA

=Engineering Controls=
An eyewash (preferably eyewash/drench hose combination unit) must be located in the immediate area. If medium-large quantities are used (>~100 ml of 100% phenol), a safety shower is also required. Contact OSEH at (734) 647-1143 for an assessment of the need for a safety shower if one is not available. Work with large open containers should be performed only in a chemical fume hood. Small amounts can be handled safely on the bench top, as long as skin contact is avoided.
�
=Work Practice Controls=
• Designate areas where phenol is stored or manipulated.

• It is highly recommended that labs using phenol (or reagents containing phenol – e.g., TRIzol) have polyethylene glycol 300 or 400 (PEG-300 or PEG-400) on hand in case of dermal exposure.
• Purchase in the smallest container that is practical for lab use.

• Purchase in a shatter-resistant container if available (such as PVC-coated glass).

• Keep containers closed as much as possible.

• Use in the smallest quantities and lowest concentration practicable for the experiment being performed.

• After work with phenol is complete, wipe down work area with soap and water solution.

=Personal Protective Equipment=
• Wear neoprene or doubled nitrile laboratory gloves, safety glasses, and lab coat. If gloves are splashed, remove them immediately and put on a new pair of gloves. If splashes may occur, wear a face shield with chemical splash goggles, and an impervious apron over the lab coat.

• If phenol and chloroform will be used together, please note that chloroform easily penetrates nitrile gloves and can degrade them, allowing phenol to contact the skin. Plan work to avoid glove contact and change gloves immediately if there is a splash. If extensive work with phenol and chloroform is done in the lab, consider the reusable ChemTek Viton/Butyl glove made for work with both of these chemicals (or they can be used for spill clean-up).

=Transportation and Storage=
• Transport phenol in secondary containment, preferably a polyethylene or other non-reactive acid/solvent bottle carrier.

• Keep container in cool, well-ventilated area.

• Keep container tightly closed and sealed until ready for use.

• Store in secondary containment away from moisture, strong oxidizers, strong caustics, plastics, rubber, nitric acid, water + heat, and chemically active metals, such as aluminum and magnesium powder, sodium, potassium, and lithium.

• Store below eye level.

• Avoid storing on the floor.

• Avoid ignition sources.

=Waste Disposal=
Handle and store hazardous waste following the guidelines above for work practice controls, transportation and storage. Because most spent, unused and expired chemicals/materials are considered hazardous wastes, they must be properly disposed of. Do not dispose of chemical wastes by dumping them down a sink, flushing in a toilet or discarding in regular trash containers, unless authorized by OSEH Hazardous Materials Management (HMM). Contact OSEH-HMM at (734) 763-4568 for waste containers, labels, manifests, waste collection and for any questions regarding proper waste disposal. Also refer to OSEH’s Hazardous Waste webpage for more information.
�
=Exposures/Unintended contact=

If the employee is in need of emergency medical attention, call 911 immediately.

For an actual chemical exposure/injury:
• Remove contaminated clothing. Flush exposed eyes or skin with water for at least 15 minutes. Seek medical attention (see below).

• If a small area of skin has been exposed to phenol, swab polyethylene glycol 300 or 400 (PEG-300 or 400) onto the affected area immediately and repeatedly until the smell of phenol is no longer evident, then seek medical attention. If PEG-300 or 400 is not available, flush area with COPIOUS amounts of water (such as from a drench hose or safety shower) for at least 15 minutes, then seek medical attention. See the US Agency for Toxic Substances and Disease Registry (ATSDR), Medical Management Guidelines for Phenol.

• For larger areas of exposed skin or eye exposure, flush area with COPIOUS amounts of water for at least 15 minutes, and then seek immediate medical attention. Please note that using high-density water irrigation will reduce phenol uptake, but if lesser amounts of water are used it will merely dilute the phenol and increase the area of exposure.

• For situations with risk of inhalation exposure (including spills of powder outside of a chemical fume hood), remove all persons from the contaminated area.

• If an ambulance is needed, call the University of Michigan Division of Public Safety and Security (DPSS) at 911 to request assistance.

Contact OSEH for advice on symptoms of chemical exposure, or assistance in performing an exposure assessment.

Report all work related accidents, injuries, illnesses or exposures to Work Connections within 24 hours by completing and submitting the Illness and Injury Report Form. Follow the directions on the Work Connections website Forms Instructions to obtain proper medical treatment and follow-up.

Complete the OSEH Laboratory Incident and Near-Miss Report form.

===TREATMENT FACILITIES:===
U-M Occupational Health Services -- Campus Employees
Mon-Fri 7:30 am - 4:30 pm

After hours - go to UM Hospital Emergency Dept. – Urgent Care Clinic
C380 Med Inn building
1500 East Medical Center Drive, Ann Arbor (734) 764-8021

University Health Services -- University students (non-life threatening conditions)
Mon-Fri 8 am – 4:30 pm, Sat 9 am – 12 pm
Contact for current hours as they may vary
207 Fletcher Street, Ann Arbor (734) 764-8320

UMHS Emergency Department -- after clinic hours or on weekends
1500 East Medical Center Drive, Ann Arbor, (734) 936-6666

�
=Spill Procedure=
• When a spill occurs, personal safety should always come first.

• Alert and clear everyone in the immediate area where the spill occurred.

• Employees in the area should be prepared to clean up minor spills, including most spills confined to the chemical fume hood. Wearing double nitrile gloves, splash goggles, face shield and lab coat (and impermeable apron, if available), use absorbent pads to absorb spilled material. After spill has been completely absorbed, wipe down contaminated area with soap and water solution. Lab personnel should take great care to avoid skin contact with phenol. If skin contact does occur, follow the instructions outlined for exposures/unintended contact in the section above.

• Contaminated PPE and clean-up materials should be placed in a clear plastic bag. Call OSEH-HMM (734) 763-4568 to arrange for waste pick-up.

• If a large spill occurs, leave the lab and call University of Michigan Division of Public Safety and Security (DPSS) at 911 to request assistance from OSEH. Do not take any action to cover the spill. Post a warning on the lab and do not allow others to enter. Have a person available that has knowledge of the incident and laboratory to assist emergency personnel.

Additional Spill Links:
• http://oseh.umich.edu/hazardous-waste/spill-response/

Report all emergencies, suspicious activity, injuries, spills, and fires to the University of Michigan Division of Public Safety and Security (DPSS) by calling 911 or texting 377911. Register with the University of Michigan Emergency Alert System via Wolverine Access.

Training of Personnel
All personnel are required to complete the General Laboratory Safety Training session (BLS025w or equivalent) via OSEH’s My LINC website. Furthermore, all personnel shall read and fully adhere to this SOP when handling phenol.

Certification
I have read and understand the above SOP. I agree to contact my Supervisor or Lab manager if I plan to modify this procedure.

*[[User:Iharvey|Iharvey]] ([[User talk:Iharvey|talk]]) 20:50, 10 November 2016 (UTC)
* [[User:Davebridges|Davebridges]] ([[User talk:Davebridges|talk]]) 21:08, 6 December 2016 (UTC)
*[[User:Reddj|Reddj]] ([[User talk:Reddj|talk]]) 14:47, 13 June 2017 (UTC)
*[[User:Cdgleason|Cdgleason]] ([[User talk:Cdgleason|talk]]) 03:55, 4 October 2018 (UTC)
*[[User:Mollyec|Mollyec]] ([[User talk:Mollyec|talk]]) 21:43, 12 September 2019 (UTC)
Laboratory Director Revision Date
�
Major Revisions (Tracking purposes only – Do not print as part of SOP)

SOP - Cryogenic Materials

2019-09-12T16:13:23Z

Mollyec: /* Certification */

[[ Category: SOP ]]
[[ Category: Lab Safety]]
=Description=
This standard operating procedure (SOP) outlines the handling and use of cryogenic materials. Review this document and supply the information required in order to make it specific to your laboratory. In accordance with this document, laboratories should use appropriate controls, personal protective equipment, and disposal techniques when handling cryogenic materials.

Cryogenic liquids have various uses in the laboratory including sample preservation, cooling of equipment, and handling and storage of biological samples.

=Potential Hazards=
Cryogenic materials are liquefied gases with boiling points less than -73°C (-100°F). They can cause tissue damage (frostbite) and asphyxiation due to oxygen displacement. They also pose a fire hazard if oxygen is condensed out of the atmosphere during the use of cryogenic liquids that have boiling points that are lower than oxygen – e.g. nitrogen, helium or hydrogen. There is a potential for explosion due to pressure buildup in closed containers. Materials intended for use with cryogenic liquids must be selected carefully as they can be weakened or fail due to embrittlement.

=Engineering Controls=
Use and store cryogens in well-ventilated areas. Closets, small rooms, and walk-in cold rooms (or similar small/unvented spaces) should be avoided to prevent buildup of the gas as the cryogen evaporates/sublimes. If using large quantities of cryogens or if there are concerns about the adequacy of ventilation, consult OSEH Research Health and Safety (734) 647-1143 to determine if additional ventilation or an oxygen sensor/alarm is needed.

Each part of a cryogenic system must have its own pressure relief system.

=Work Practice Controls=
• Cryogenic gases can condense oxygen out of the air, creating a localized oxygen enriched environment. Therefore they must be used and stored away from flammable materials, open flames and ignition sources.

• Do not put your head inside a liquid nitrogen freezer or other enclosed space containing a cryogen. Never allow people to travel in an elevator with cryogenic liquids.

• Do not use hollow rods or tubes as dipsticks. (When a warm tube is inserted into a cryogen, liquid will spout from the top of the tube.

• When retrieving cryopreservation vials that have been stored in liquid nitrogen, raise the vials out of the liquid phase and store them in the gas above the liquid for 24 hours to allow any trapped liquid nitrogen to escape. After this period, the vials may be removed. Retrieving these vials directly from the liquid phase may cause the vials to rupture if liquid nitrogen has become trapped inside. This may expose the employee to flying bits of plastic, biological specimens or cryoprotective agents (such as dimethyl sulfoxide) that were in the vial.

• Check the liquid levels of Dewars regularly. If the liquid evaporates more rapidly than normal, the Dewar may be losing its vacuum.

• Also follow the SOP for asphyxiants.

=Personal Protective Equipment (PPE)=
• Wear loose fitting cryogenic gloves, safety goggles, face shield, lab coat, long pants and closed toe shoes when handling and transporting cryogenic materials.

• Lab coats should be knee length and have no pockets or cuffs to catch the liquid.

• Pants should not have cuffs and should be long enough to go over the shoe or boot.

=Transportation and Storage=
• Never store cryogenic liquids or dry ice in walk-in cold rooms.

• Use and store liquid hydrogen and helium away from flammable materials and ignition sources. (These gases can condense oxygen out of the air, creating a localized oxygen enriched environment.)

• For liquid helium and hydrogen storage systems, check the pressure relief and inspect the system for leaks regularly.

• These gases are cold enough to solidify atmospheric air; leaks in storage systems for these gases may become plugged with solidified air. If the pressure relief device becomes plugged, the container may over-pressurize and fail.

• Use and store liquid oxygen away from open flames. Post a “No Open Flames” sign in liquid oxygen storage areas.

• Store and transport cryogenic materials only in Dewars or cryogenic liquid cylinders designed for that particular cryogen. Inspect Dewars daily to insure that no air or ice plugs exist in the neck openings.

• Cryogens may be transported in elevators only in containers certified to leak at less than or equal to 1 liter of liquid (or 1 kilogram of solid) per day. Never allow passengers to travel in an elevator with cryogenic liquids.

=Waste Disposal=
If a cryogen (such as dry ice) has been mixed with a liquid that would normally be disposed of as hazardous waste (such as alcohol, as in a cold trap), follow disposal procedures for that liquid. • Cryogens must not be dumped into sinks, as the thermal shock is likely to damage the sink and/or the plumbing.

• As the unneeded cryogen is evaporating, good ventilation is essential to prevent hazardous air concentrations of the gas.

• Do not put unneeded cryogen in a sealed container, as the build-up of pressure could cause an explosion.

=Exposures/Unintended Contact=

If the employee is in need of emergency medical attention, call 911 immediately.

For an actual chemical exposure/injury:
• Refer to SDS.

• Flush exposed eyes or skin with water with warm water for at least 15 minutes, then seek medical attention (see below).

• Do not rub affected area.

Contact OSEH for advice on symptoms of chemical exposure, or assistance in performing an exposure assessment.

Report all work related accidents, injuries, illnesses or exposures to Work Connections within 24 hours by completing and submitting the Illness and Injury Report Form. Follow the directions on the Work Connections website Forms Instructions to obtain proper medical treatment and follow-up.

Complete the OSEH Laboratory Incident and Near-Miss Report form.

===TREATMENT FACILITIES:===
U-M Occupational Health Services -- Campus Employees
Mon-Fri 7:30 am - 4:30 pm

After hours - go to UM Hospital Emergency Dept. – Urgent Care Clinic
C380 Med Inn building
1500 East Medical Center Drive, Ann Arbor (734) 764-8021

University Health Services -- University students (non-life threatening conditions)
Mon-Fri 8 am – 4:30 pm, Sat 9 am – 12 pm
Contact for current hours as they may vary
207 Fletcher Street, Ann Arbor (734) 764-8320

UMHS Emergency Department -- after clinic hours or on weekends
1500 East Medical Center Drive, Ann Arbor, (734) 936-6666

=Spill Procedure=
• When a spill occurs, personal safety should always come first.

• Alert and clear everyone in the immediate area where the spill occurred.

Spills of cryogenic liquid can cause depletion of oxygen in the area. For spills over 1 liter (or smaller amounts in very small rooms), leave the area immediately and call the University of Michigan Division of Public Safety and Security (DPSS) at 911.
Additional Spill Links:
• http://oseh.umich.edu/hazardous-waste/spill-response/

Report all emergencies, suspicious activity, injuries, spills, and fires to the University of Michigan Division of Public Safety and Security (DPSS) by calling 911 or texting 377911. Register with the University of Michigan Emergency Alert System via Wolverine Access.

=Training of Personnel=
All personnel are required to complete the General Laboratory Safety Training session (BLS025w or equivalent) via OSEH’s My LINC website. Furthermore, all personnel shall read and fully adhere to this SOP when handling cryogenic materials.

=Certification=
I have read and understand the above SOP. I agree to contact my Supervisor or Lab manager if I plan to modify this procedure.

*[[User:Iharvey|Iharvey]] ([[User talk:Iharvey|talk]]) 20:44, 10 November 2016 (UTC)
*[[User:Elhabbal|Elhabbal]] ([[User talk:Elhabbal|talk]]) 22:11, 12 June 2017 (UTC)
*[[User:Reddj|Reddj]] ([[User talk:Reddj|talk]])
*[[User:Mollyec|Mollyec]] ([[User talk:Mollyec|talk]]) 21:43, 12 September 2019 (UTC)
Prior Approval required – Is this procedure hazardous enough to warrant prior approval from the Laboratory Director? ☐ YES ☐ NO

Laboratory Director Revision Date
�
Major Revisions (Tracking purposes only – Do not print as part of SOP)

SOP - B-mercaptoethanol

2019-09-12T16:12:07Z

Mollyec: /* Certification */

[[ Category: SOP ]]
[[ Category: Lab Safety]]
=Description=
This standard operating procedure (SOP) outlines the handling and use of B-mercaptoethanol (BME). Review this document and supply the information required in order to make it specific to your laboratory. In accordance with this document, laboratories should use appropriate controls, personal protective equipment, and disposal techniques when handling BME.

BME is used as an enzyme reactivator in inhibited systems, a reducing agent in the fluorescent reaction of o-phthaldialdehyde, and amino-acids in alkaline media and is also used to dissociate proteins.

=Potential Hazards=
• BME has a very low odor threshold (0.12-0.64 ppm) and smells similar to the odorant used in natural gas. If the odor becomes widespread, people in nearby areas may suspect a natural gas leak. This can lead to calls to the fire department and/or evacuation of the building, which can be inconvenient and disruptive.

• BME can be toxic if ingested and fatal if inhaled or absorbed through the skin.

• Vapors can irritate the eyes, mucous membranes, and respiratory tract. Symptoms of inhalation exposure may include coughing, sore throat, and/or shortness of breath.

• When BME is heated to decomposition, toxic fumes including sulfur oxides and carbon oxides will be emitted.

• BME is combustible as a liquid or vapor.

• Reactions of BME with strong acids or alkali metals will release flammable hydrogen gas.

=Engineering Controls=
ALWAYS work with BME inside a chemical fume hood or 100% exhausted biological safety cabinet (Class II, Type B2).

=Work Practice Controls=
• BME is incompatible with metals, oxidizing agents, acids, alkalis, calcium hypochlorite, aliphatic amines, and isocyanates.

• Purchase and use in the smallest practical quantities for the experiment being performed.

• Know the location of the nearest fire extinguisher before beginning work.

• Eliminate ignition sources such as open flames and hot surfaces.

• Keep containers closed as much as possible when not in use.

• Be aware of skin absorption as a possible route of exposure. Plan work so that minimal glove contact is expected, and purchase appropriate gloves for cleaning up small spills.

• If glove contact occurs, change gloves immediately.

=Personal Protective Equipment (PPE)=
At a minimum, double-glove using nitrile laboratory gloves and wear a lab coat and safety glasses when working with BME. If there is a possibility of splashing, wear chemical splash goggles and/or a face shield.

=Transportation and Storage=
• Do not store near sources of ignition, oxidizing agents, acids, alkaline compounds, or any other incompatible materials.

• Store BME in a sealed secondary container in a well-ventilated area.

• The container must be tightly closed, resealed, and stored upright to avoid leakage.

• Avoid storing on the floor.

• Transport toxic liquids in secondary containment, preferably a polyethylene or other non-reactive acid/solvent bottle carrier.

• Suitable fire control devices (such as fire extinguishers) must be available at locations where flammable or combustible liquids are stored.

=Waste Disposal=
Handle and store hazardous waste following the guidelines above for work practice controls, transportation and storage. Contact OSEH Hazardous Materials Management at (734) 763-4568 for waste containers, labels, manifests, waste collection and for any questions regarding proper waste disposal. Also refer to OSEH’s Hazardous Waste webpage for more information.

=Exposures/Unintended Contact=
If the employee is in need of emergency medical attention, call 911 immediately.

==For an actual chemical exposure/injury:==
• In case of skin contact: Flush the skin with copious amounts of water for at least 15 minutes, and then seek medical attention (see below).

• In case of eye contact: Flush contaminated eye(s) immediately with copious amounts of water for at least 15 minutes, and then seek medical attention (see below).

• In case of inhalation: Assist conscious persons to an area with fresh, uncontaminated air, and then seek medical attention (see below).

Contact OSEH for advice on symptoms of chemical exposure, or assistance in performing an exposure assessment.

Report all work related accidents, injuries, illnesses or exposures to Work Connections within 24 hours by completing and submitting the Illness and Injury Report Form. Follow the directions on the Work Connections website Forms Instructions to obtain proper medical treatment and follow-up.

Complete the OSEH Laboratory Incident and Near-Miss Report form.

===TREATMENT FACILITIES:===

U-M Occupational Health Services -- Campus Employees
Mon-Fri 7:30 am - 4:30 pm

After hours - go to UM Hospital Emergency Dept. – Urgent Care Clinic
C380 Med Inn building
1500 East Medical Center Drive, Ann Arbor (734) 764-8021

University Health Services -- University students (non-life threatening conditions)
Mon-Fri 8 am – 4:30 pm, Sat 9 am – 12 pm

Contact for current hours as they may vary
207 Fletcher Street, Ann Arbor (734) 764-8320

UMHS Emergency Department -- after clinic hours or on weekends
1500 East Medical Center Drive, Ann Arbor, (734) 936-6666

==Spill Procedure==
• When a spill occurs, personal safety should always come first.

• Alert and clear everyone in the immediate area where the spill occurred.

Employees in the area should be prepared to clean up minor spills confined to the chemical fume hood. Wearing appropriate gloves (e.g., butyl rubber, Silver Shield®, nitrile), splash goggles, lab coat (and impermeable apron, if available), use an inert absorbent material (sorbent pads, vermiculite, dry sand) to clean up the spill. Do not use combustible materials (i.e. saw dust) to absorb spill. Contaminated PPE and clean-up materials must be placed in a sealed container for pick-up by OSEH-HMM (see waste disposal section for more information).

For large spills, or any spills of BME outside of the fume hood, contact the University of Michigan Division of Public Safety and Security (DPSS) by calling 911.
Additional Spill Links:

• http://oseh.umich.edu/hazardous-waste/spill-response/

Report all emergencies, suspicious activity, injuries, spills, and fires to the University of Michigan Division of Public Safety and Security (DPSS) by calling 911 or texting 377911. Register with the University of Michigan Emergency Alert System via Wolverine Access.

=Training of Personnel=
All personnel are required to complete the General Laboratory Safety Training session (BLS025w or equivalent) via OSEH’s My LINC website. Furthermore, all personnel shall read and fully adhere to this SOP when handling BME.

=Certification=
I have read and understand the above SOP. I agree to contact my Supervisor or Lab manager if I plan to modify this procedure.

*[[User:Iharvey|Iharvey]] ([[User talk:Iharvey|talk]]) 19:42, 10 November 2016 (UTC)
*[[User:Elhabbal|Elhabbal]] ([[User talk:Elhabbal|talk]]) 21:43, 12 June 2017 (UTC)
*[[User:Reddj|Reddj]] ([[User talk:Reddj|talk]])
*[[User:Mollyec|Mollyec]] ([[User talk:Mollyec|talk]]) 21:43, 12 September 2019 (UTC)
Prior Approval required – Is this procedure hazardous enough to warrant prior approval from the Laboratory Director? ☐ YES ☐ NO
Laboratory Director Revision Date
�
Major Revisions (Tracking purposes only – Do not print as part of SOP)

Assessing isoproterenol-stimulated whole-body lipolysis in vivo

2019-03-01T16:52:56Z

Mollyec: /* Glycerol and Triglyceride determination from serum (use Sigma Diagnostics Triglyceride Assay Kit) */

__NOTOC__
[[ Category: Mouse Work ]]
[[ Category: Lipolysis ]]
[[ Category: Lipid Metabolism ]]

== Background ==
Lipolysis is the hydrolysis of triglycerides into glycerol and free fatty acids. Lipolysis is induced through activation of beta adrenergic receptors. Isoproterenol/Isoprenaline is a non-selective beta adrenergic agonist structurally similar to adrenaline. By administering isoproterenol ''in vivo'', it is possible to artificially stimulate whole-body lipolysis and assess changes in the concentrations of the products of lipolysis (i.e., glycerol and free fatty acids) in the blood.

==Reagents Needed==
#(−)-Isoproterenol hydrochloride (Sigma cat# I6504)
#PBS
#Isofluorane

== Experimental protocol ==
Note: Mice do not need to be in a fasted state prior to this test.
#Briefly anesthetize mice with isofluorane and collect blood via retro orbital bleed. Allow blood to clot over ice.
#Inject 10 mg/kg isoproterenol (prepared fresh in sterile PBS) via intraperitoneal injection. This will be 10mg in 10mL then inject 10uL/g mouse.
#Wait 15 minutes.
#Briefly anesthetize mice with isoflurane and collect blood via retro orbital bleed. Allow blood to clot over ice. ***Note: Take care when anesthetizing mice the second time. They will go under VERY quickly, so watch the mouse closely***
#Centrifuge blood samples and collect serum.
#Assay serum for NEFA's and Glycerol/Triglycerides using Wako and Sigma assay kits, respectively.

== NEFA determination from serum (use Wako Diagnostics NEFA-HR(2) Microtiter Kit) ==
*Note: The manufacturers protocol says to use 225 uL of reaction buffer A and 75 uL of reaction buffer B with 4 uL of sample, read at 560:670 nm. This uses more reagent than is necessary for the volume of serum/concentrations of NEFA that we would typically analyze.

#To wells of a clear 96 well plate, add 4uL of water (as blank), appropriate standard (the kit comes with stock of 1 mEq/L. Dilute the stock in ddH20 to give 0.25 and 0.5 mEq/L standards. For the high standard, add 8 uL of the 1 mEq/L stock. Due to the extra volume, the concentration of this last standard is actually 1.97 mEq/L).
#To remaining wells, add 4 uL of serum samples. ***Note: You may need to add more/less serum, depending on the NEFA concentration of your samples. The assay is linear between 0.01-4.00 mEq/L***
#To each well, add 100 uL of reaction buffer A, mix gently and incubate at 37 deg C for ~5 minutes.
#Allow plate to return to room temperature before reading at 560:660 nm. These are the initial readings.
##Note: If the plate reader being used does not allow for measuring two wavelengths simultaneously, read the plate at 560 nm. The rationale behind measuring two wavelengths simultaneously is to account for possible sample contamination by products of haemolysis, which can interfere with the assay.
#To each well, add 50 uL of reaction buffer B and incubate at 37 deg C for ~5 minutes. The wells will turn purple. If they do not, check whether the reagents are within the best before date.
#Allow plate to return to room temperature before re-reading at 560:660 nm. These are the final readings.
#Calculations
##For both initial and final readings, subtract the absorbence values obtained at the 670 nm wavelength from those obtained at the 560 nm wavelength.
##Subtract the initial absorbance reading from the final reading, then calculate sample concentrations from the standard curve.

== Glycerol and Triglyceride determination from serum (use Sigma Diagnostics Triglyceride Assay Kit) ==
#To wells of a clear 96 well plate, add 3 uL of water (as blank), the appropriate volume of standard (the kit comes with a stock standard of 2.5 mg/mL triolein; to replicate wells, add 1, 2, 3 and 4 uL of this stock. The assay is only linear up to concentrations of 10 mg/mL, which is 4 uL of 2.5 ug/mL standard).
#To each well, add 80 uL of glycerol reagent.
#To each well, add 3-8 uL of serum samples (3 if you expect a lot of lipolysis, and greater volume if you expect less).
#Incubate plate at 37 deg C for 5 minutes.
#Read the plate at 540 nm. This is the initial reading.
#To each well, add 20 uL of triglyceride reagent. Incubate plate at 37 deg C for ~5 minutes. The triglyceride reagent contains a lipase that breaks down the triglycerides into glycerol and fatty acids. The assay measures the glycerol in the sample, so the first reading tells us how much glycerol is present.
#Re-read the plate at 540 nm. This is the final reading. The second reading tells us how much glycerol is present after the triglycerides present in the serum have been broken down.
#Calculations
##Subtract the initial reading from the final reading. This tells us what proportion of the glycerol in the sample came from triglycerides.
##For each of the initial, final and calculated absorbances, calculate the concentration of the samples from the standard curve.

Western Blotting

2019-01-07T18:07:27Z

Mollyec: /* Protocol */

==Materials==
*Transfer Buffer (200 mL Methanol, 100 mL 10X [[ Transfer Buffer ]] to final 1L volume)
*Transfer Apparatus, either Bio-Rad or Invitrogen

==Protocol==
#Run SDS-PAGE gel using [[ SDS-PAGE Running Buffer ]] and prepare diluted transfer buffer
## Use a prepared 5-12% tris gel (in the 4 degree). Remove it from the packaging, remove the white strip of tape from the bottom back, and gently pull the comb out and rinse with water.
##Load into gel tank. Fill with SDS page Running buffer (1X) to the fill line in the front and halfway up the back
##Load 3 microliters of protein ladder (purple), and 10 microliters of each sample into separate wells.
## Place top on tank, plug into power source and run at 125 Volts until samples and ladder reach the bottom of the gel
#Make sandwich (black side, sponge, filter paper, gel, nitrocellulose, filter paper, sponge, clear side), ensuring no bubbles between layers with black piece on bottom and layer as above. Place in apparatus so that the black sandwich touches the black transfer piece. Fill with transfer buffer.
#Transfer 4h at 75V (in cold room) or overnight at 35V (room temp).
#Stain for total protein with Revert total protein stain on rocker for 5 minutes --when finished pour total protein stain back in bottle for later use!
#Rinse twice in revert wash solution (60ml MeOH, 13.4 ml Aceditc Acid, 126.6 ml Water)
#Scan using licor for total protein, which will be used to normalize the blot
#Rinse nitrocellulose in revert reversal solution for at least 5 and no more than 10 minutes until nitrocellulose appears clear again (.2g NaOH, 60ml MeOH, 140ml Water)
#Rinse nitrocellulose in 2% BSA (2g BSA in 100ml TBST, stored in fridge) for 1 hour
#Incubate with primary antibody (check for dilution) in 2% BSA for >1h
#Wash blot every 5 minutes for 15 min with TBST.
#Incubate with appropriate secondary antibody (10 000X) for 45min-1h (20 ml 2% BSA 1ul of both secondary antibodies, all found in fridge Ab stocks with blue dots on top)
#Wash blot every 5 minutes for 15 min with TBST.
#Rinse once or twice with double distilled water
#Prepare ECL by mixing one volume of the black bottle solution with one volume of the white bottle solution (be careful not to cross-contaminate ECL bottles with wrong solution).
#Drain excess buffer from blot and cover with ECL for about a minute
#Drain excess ECL from blot, cover with saran wrap and expose film

==If Using LiCor==
#Start -> New -> Scan Image -> Login -> Peloquin -> Password Located in Desk -> Select Dimensions -> Start Scan

[[ Category: Western Blotting ]]

Western Blotting

2019-01-07T18:07:05Z

Mollyec: /* Protocol */

==Materials==
*Transfer Buffer (200 mL Methanol, 100 mL 10X [[ Transfer Buffer ]] to final 1L volume)
*Transfer Apparatus, either Bio-Rad or Invitrogen

==Protocol==
#Run SDS-PAGE gel using [[ SDS-PAGE Running Buffer ]] and prepare diluted transfer buffer
## Use a prepared 5-12% tris gel (in the 4 degree). Remove it from the packaging, remove the white strip of tape from the bottom back, and gently pull the comb out and rinse with water.
##Load into gel tank. Fill with SDS page Running buffer (1X) to the fill line in the front and halfway up the back
##Load 3 microliters of protein ladder (purple), and 10 microliters of each sample into separate wells.
## Place top on tank, plug into power source and run at 125 Volts until samples and ladder reach the bottom of the gel
#Make sandwich (black side, sponge, filter paper, Gel, Nitrocellulose, Filter paper, Sponge, clear side), ensuring no bubbles between layers with black piece on bottom and layer as above. Place in apparatus so that the black sandwich touches the black transfer piece. Fill with transfer buffer.
#Transfer 4h at 75V (in cold room) or overnight at 35V (room temp).
#Stain for total protein with Revert total protein stain on rocker for 5 minutes --when finished pour total protein stain back in bottle for later use!
#Rinse twice in revert wash solution (60ml MeOH, 13.4 ml Aceditc Acid, 126.6 ml Water)
#Scan using licor for total protein, which will be used to normalize the blot
#Rinse nitrocellulose in revert reversal solution for at least 5 and no more than 10 minutes until nitrocellulose appears clear again (.2g NaOH, 60ml MeOH, 140ml Water)
#Rinse nitrocellulose in 2% BSA (2g BSA in 100ml TBST, stored in fridge) for 1 hour
#Incubate with primary antibody (check for dilution) in 2% BSA for >1h
#Wash blot every 5 minutes for 15 min with TBST.
#Incubate with appropriate secondary antibody (10 000X) for 45min-1h (20 ml 2% BSA 1ul of both secondary antibodies, all found in fridge Ab stocks with blue dots on top)
#Wash blot every 5 minutes for 15 min with TBST.
#Rinse once or twice with double distilled water
#Prepare ECL by mixing one volume of the black bottle solution with one volume of the white bottle solution (be careful not to cross-contaminate ECL bottles with wrong solution).
#Drain excess buffer from blot and cover with ECL for about a minute
#Drain excess ECL from blot, cover with saran wrap and expose film

==If Using LiCor==
#Start -> New -> Scan Image -> Login -> Peloquin -> Password Located in Desk -> Select Dimensions -> Start Scan

[[ Category: Western Blotting ]]

QPCR

2018-10-23T15:43:41Z

Mollyec: /* Plate Preparation */

__NOTOC__
==Real Time qPCR==
===Materials===
*cDNA: see [[First_Strand_cDNA_Synthesis_(AB_Kit)|First Strand cDNA Synthesis (AB Kit)]] for details
*SYBR Green PCR Master Mix Applied Biosystems (ThermoFisher Catalog # 4367659; [https://www.thermofisher.com/order/catalog/product/4367659 Vendor Link])
*384 well qPCR plate (ThermoFisher Catalog # 4309849) and covers (Catalog # 4360954)
*Primers (Dilute to 0.4 uM mixture of fwd and rev. From 100 uM stocks- the 100uM stock is prepared by adding 227 uL of distilled water to 22.7nmol of a gene as an example- this is 4uL Forward Primer, 4 uL Reverse Primer and 992 uL Water). This primer mix can be stored at -20 in a Working Primers box. Design primers according to [[Primer Design for qPCR]]

===Plate Preparation===
#Book 2h on qPCR machine by signing up on the sheet in room 7013
#Prepare cDNA and dilute in water in a 96 well plate. Typically a 20x dilution of cDNA leaves enough to be detected.
#Get optically clear 384 well plate and keep on paper towel. Do not touch bottom of plate.
#Sketch out the plate in your notes. Typically rows are different primers while columns are different cDNA's
#Calculate how many samples x how many replicates/per sample (start with 3 or 4 until you are consistent enough technically to decrease). This will be the number of wells need for each primer.
#Prepare a Primer/SYBR Green mixture for each primer. For each well you will need 5 uL SYBR green + 2.5 uL Primer working stock, so if you have calculated you need 10 wells per primer that is 50 uL SYBR Green + 25 uL Primers. Make up 10-20% more than you need.
#Using the repeater multichannel pipette put on 2 or 3 tips (depending on your plate arrangement) and set to aspirate however many samples you have and dispense 7.5 uL per well.
#Dispense 7.5 uL of Primer/SYBR mixture into each well, dispensing at the bottom of the well.
#Using the ClipTip multichannel add 2.5 uL of cDNA to each applicable well. You don't need to change tips between wells.
#Once the plate is completed, carefully put an optically clear cover on it using the plastic square to ensure the edges are sealed, being very careful not to leave fingerprints on the seal.
#You can prepare the plate ~3h beforehand, keeping it at 4C until the machine is ready.
#Immediately before the run spin the plate briefly (2 mins at 4000 RPM) in the swinging bucket centrifuge.

===Run Protocol===
* Set up run using [[Set up qPCR Run on Thermo Cloud and QuantStudio|Thermo Cloud/QuantStudio]] or [[Set up qPCR Run on Roche LightCycler|Roche LightCycler]]

==Calculations==
see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC55695 for considerations on calculations

[[ Category: Molecular Biology ]]
[[ Category: RNA ]]
[[ Category: qPCR ]]
[[ Category: Transcription ]]
[[ Category: Expression ]]

Preparation of RNA Samples from Mouse Tissues

2018-10-22T20:51:22Z

Mollyec: /* Materials */

==Safety Information==
* [[SOP-_Phenol|SOP - Phenol]]
* [[SOP_-_Chloroform|SOP - Chloroform]]

==Materials==
*PureLink RNA Mini Kit (Invitrogen cat#12183-018A)
*Mouse Tissue (50-100 mg, about a 3mm cube)
*TRIZol (Invitrogen cat# 12183-555)
*Chloroform (in solvent cabinet)
*Label tubes, for each sample need: 2.0 mL tube (to cut sample into), 1.5 mL tubes (X2), 1 spin column*, 1 recovery tube*, 1 collection tube* (*= in the PureLink kit)
*70% Ethanol make with RNAase free water and 100% Ethanol.

==Protocol==
#Cut ~50-100 mg of tissue. If tissue is in RNAlater, cut at room temperature. If tissue is frozen, cut on dry ice. Weigh into a fresh 2mL eppendorf tube and keep on ice.
#Add 1 mL TRIzol reagent to each 2 mL tube.
#Add 1 ball bearing to each tube.
#Using tissue grinder, homogenize tissue for 3 minutes at 25 Hz (make sure there are no remaining clumps, maybe take longer for muscle). If chunks remain after 3 minutes, homogenize until chunks are gone in 2 minute increments.
#Incubate 5 minutes at room temperature.
#Transfer sample to a new 1.5 vial. The ball bearings are reusable after cleaning so do not discard them.
#Add 200 uL Chloroform and shake vigourously by hand for 15 seconds. Do NOT vortex.
#Incubate at room temperature for 2-3 minutes.
#Centrifuge in a cold eppendorf centrifuge (4C) on maximum for 15 minutes. Sample will separate into a lower, red chloroform phase, an interphase and a colorless upper phase which contains the RNA. (WAT tissue may separate into 3 phases, RNA is the top, colorless phase.
#Add 400 uL of 70% ethanol to a fresh tube.
#Carefully transfer 400 uL of the upper phase to the ethanol tube and mix by vortexing. The remaining chloroform in the previous vial should be disposed of in the phenol waste container under the hood.
#Remove 700 uL of ethanol/upper phase mixture (invert briefly to mix before taking sample) and add to a spin column in a collection tube.
#Spin 15 seconds on max. Discard flow through. Add remaining sample, respin and discard flow through.
#Add 700 uL Wash Buffer I to spin column.
#Spin 15 seconds on max. Discard flow through and collection tube, add a new collection tube.
#Add 500 uL Wash Buffer II (make sure ethanol was added to the wash buffer) to the spin cartridge.
#Spin 15 seconds on max. Discard the flow through and keep the same collection tube.
#Repeat wash by adding 500 uL Wash Buffer II to the spin cartridge.
#Spin 15 seconds on max. Discard the flow through and keep the same collection tube.
#Spin 1 minute on max to dry the cartridge. Discard the collection tube and place spin column into a clean recovery tube. Add 100 uL RNAase free water to the center of each tube. This can be adjusted to between 30 and 300 uL elution buffer if necessary.
#Incubate at room temperature for 1 minute.
#Spin 2 minutes at 12500 rpm to get purified RNA.
#Quantify the RNA using the nanodrop.

[[Category:qPCR]]
[[Category:Expression]]
[[Category:Transcription]]
[[Category:RNA]]
[[Category:Mouse Work]]
[[Category:Tissues]]

Preparation of RNA Samples from Mouse Tissues

2018-10-22T20:48:53Z

Mollyec: /* Protocol */

==Safety Information==
* [[SOP-_Phenol|SOP - Phenol]]
* [[SOP_-_Chloroform|SOP - Chloroform]]

==Materials==
*PureLink RNA Mini Kit (Invitrogen cat#12183-018A)
*Mouse Tissue (50-100 mg, about a 3mm cube)
*TRIZol (Invitrogen cat# 12183-555)
*Chloroform (in solvent cabinet)
*Label tubes, for each sample need 3 eppies, at least one of which is 2 mL and one of which is a recovery tube from the PureLink kit.
*70% Ethanol make with RNAase free water and 100% Ethanol.

==Protocol==
#Cut ~50-100 mg of tissue. If tissue is in RNAlater, cut at room temperature. If tissue is frozen, cut on dry ice. Weigh into a fresh 2mL eppendorf tube and keep on ice.
#Add 1 mL TRIzol reagent to each 2 mL tube.
#Add 1 ball bearing to each tube.
#Using tissue grinder, homogenize tissue for 3 minutes at 25 Hz (make sure there are no remaining clumps, maybe take longer for muscle). If chunks remain after 3 minutes, homogenize until chunks are gone in 2 minute increments.
#Incubate 5 minutes at room temperature.
#Transfer sample to a new 1.5 vial. The ball bearings are reusable after cleaning so do not discard them.
#Add 200 uL Chloroform and shake vigourously by hand for 15 seconds. Do NOT vortex.
#Incubate at room temperature for 2-3 minutes.
#Centrifuge in a cold eppendorf centrifuge (4C) on maximum for 15 minutes. Sample will separate into a lower, red chloroform phase, an interphase and a colorless upper phase which contains the RNA. (WAT tissue may separate into 3 phases, RNA is the top, colorless phase.
#Add 400 uL of 70% ethanol to a fresh tube.
#Carefully transfer 400 uL of the upper phase to the ethanol tube and mix by vortexing. The remaining chloroform in the previous vial should be disposed of in the phenol waste container under the hood.
#Remove 700 uL of ethanol/upper phase mixture (invert briefly to mix before taking sample) and add to a spin column in a collection tube.
#Spin 15 seconds on max. Discard flow through. Add remaining sample, respin and discard flow through.
#Add 700 uL Wash Buffer I to spin column.
#Spin 15 seconds on max. Discard flow through and collection tube, add a new collection tube.
#Add 500 uL Wash Buffer II (make sure ethanol was added to the wash buffer) to the spin cartridge.
#Spin 15 seconds on max. Discard the flow through and keep the same collection tube.
#Repeat wash by adding 500 uL Wash Buffer II to the spin cartridge.
#Spin 15 seconds on max. Discard the flow through and keep the same collection tube.
#Spin 1 minute on max to dry the cartridge. Discard the collection tube and place spin column into a clean recovery tube. Add 100 uL RNAase free water to the center of each tube. This can be adjusted to between 30 and 300 uL elution buffer if necessary.
#Incubate at room temperature for 1 minute.
#Spin 2 minutes at 12500 rpm to get purified RNA.
#Quantify the RNA using the nanodrop.

[[Category:qPCR]]
[[Category:Expression]]
[[Category:Transcription]]
[[Category:RNA]]
[[Category:Mouse Work]]
[[Category:Tissues]]

Primer Design for qPCR

2017-06-29T17:48:43Z

Mollyec: /* Making Your Own Primers From Entrez */

==Finding Known Primer Sets==
* Check papers, especially supplementary tables
* Check PrimerBank http://pga.mgh.harvard.edu/primerbank/index.html

==Making Your Own Primers From Entrez==
# Check if the primer is available in the lab using the Primer Data sheet at https://docs.google.com/spreadsheets/d/1kQ1G52XuoqpAWHIWT5H2UDz4aDhP285oUyYcj7XTB24/edit?ts=5925e7a2#gid=0. If the primers are not available, proceed to the next steps
#Find your RNA sequence on entrez by starting with the gene at http://www.ncbi.nlm.nih.gov/gene (make sure its the correct species). Then pick the mRNA you want to probe, based on the isoform structure on that page. Click on that nucleotide. (it will be an NM number)
# Under analyse this sequence click '''Pick Primers'''
# Under PCR Product Size pick 70-150 as the range
# Under Exon/intron selection -> Intron Inclusion check the box by '''Primer pair must be separated by at least one intron on the corresponding genomic DNA'''
# If you want to look at a specific region of the mRNA and an appropriate primer pair in that region is not selected, change the range under PCR template on the top
# Click '''Get Primers''' on the bottom and wait for results
# Print this out, for future reference and when you order primers name them as follows species-Gene-direction-seq.start-seq.end, for example '''mm-SREBF1-FWD-127-254'''.
# Enter both primers in the Primer Data sheet used in Step 1

[[ Category: qPCR ]]
[[ Category: mRNA ]]
[[ Category: Transcription ]]
[[ Category: Molecular Biology ]]

Glycogen Determination from Tissues

2017-06-15T16:55:16Z

Mollyec: /* Protocol */

[[Category:Glycogen]]
[[Category:Metabolism]]
[[Category:Mouse Tissues]]
[[Category:Mouse Work]]

==Materials and Buffers==
* Screw Capped Vials
* 30% KOH, prepared fresh
* 1M Sodium Sulfate
* Ethanol
* 50 mM Sodium Acetate, pH 4.8
* Amyloglucosidase 0.3 mg/mL in 50 mM Sodium Acetate. Stored in -80. (Sigma A7420-5MG)
* Glucose quantification kit (Wako Autokit Glucose Buffer Solution cat # 439-9091; for protocol see http://www.wakodiagnostics.com/pi/pi_autokit_glucose.pdf)
* Glucose standard solution (200 or 500 mg/dL; Wako)

==Protocol==
# Weight out 30-90 mg tissue into a '''screw cap vial''' and record weights. Screw cap vials are really important or else the lids will pop off.
# Turn on the heating block and set it to 95C (this can take up to 15 minutes to reach the desired temperature).
# Add 300 uL 30% KOH and place on 95C heat block for 30 min with occasional mixing by gently tapping the vials to ensue the tissue dissolves completely. Make sure all the sample is initially immersed in KOH.
# Cool and then add 100 uL Sodium Sulfate and 800 uL Ethanol.
# Boil for 5 min.
# Turn on the incubator and set it to 37C.
# Centrifuge at 13 000 RPM for 5 min.
# Aspirate the solution leaving the pellet at the bottom of the vial.
# Resuspend pellet in 200 uL water while making sure that all the glycogen dissolves in water, then add 400 uL ethanol.
# Boil 5 min, spin 5 min and Repeat wash steps twice more (Wash Steps: Aspirate -> Resuspend with H20 and EtOH -> Boil -> Centrifuge -> Aspirate).
# Dry pellet on the bench by leaving the vial cap open.
# Prepare amyloglucosidase solution by diluting the AG stock 100X into 50 mM Sodium Acetate, pH 4.8. Prepare enough for 200 uL per tube plus some extras
# Resuspend pellets in 200uL amyloglucosidase solution and incubate at 37C for 3h-O/N
# Quantify glucose using kit:
## Add 100 uL of Glucose buffer solution to each well, including wells for standard curve and blank
## Add 1-5 ul of glucose standard 200mg/dL diluted 1:5
## Add 10 uL digested glycogen for fasted samples, and use a 10X dilution for refed tissues (using either 5 or 2ul from the diluted sample)
## Mix and incubate at 37C for 5 min
## Measure absorbance at 505 nm
## Calculate glycogen levels as umoles glucose released per g tissue (should be ~10)

==Calculations==
# Use this Sweave template: https://raw.github.com/davebridges/biomolecule-scripts/master/R/Sweave/glycogen-analysis-tissue.Rnw
Reference:

PMID 15282316

SOP - Bleach

2017-06-13T14:52:17Z

Mollyec: /* Certification */

[[ Category: SOP ]]
[[ Category: Lab Safety ]]

==Description==
This standard operating procedure outlines the handling and use of bleach. Review this document and supply the information required in order to make it specific to your laboratory. In accordance with this document, laboratories should use appropriate controls, personal protective equipment, and disposal techniques when handling bleach.
This document is modified from http://www.oseh.umich.edu/docs/SOP/Bleach.docx

Bleach is a general term that typically refers to an aqueous mixture that has a primary active ingredient of either Sodium Hypochlorite or Calcium Hypochlorite and usually at concentrations of 3 - 10%. Bleach is used primarily as an oxidizing, cleaning, or a bleaching agent, as a disinfectant or within drinking water and waste water purification systems.

Synonyms of bleach include sodium hypochlorite, Clorox, liquid bleach, antiformin, chlorox, Carrel-dakin solution, Chloros, Dakin’s solution, hychlorite, Javelle water.

==Useful Bleach Links==
* http://www.atsdr.cdc.gov/toxfaqs/tfacts184.pdf
* http://www.atsdr.cdc.gov/Mhmi/mmg184.pdf
* http://www.asc.co.id/uplimg/File/03%20zMSDS_NaClO_ASC%20R3.pdf
* http://www.cl2.com/documents/Incompatibility%20Chart.pdf

==Potential Hazards==
* Corrosive. May cause severe irritation or damage to eyes or skin. Vapor may irritate eyes and respiratory tract.
* Incompatible with many chemicals/agents. Hazardous gases (including chlorine and chloramines) may be released if bleach is mixed with an incompatible material.
* Exposure to these gases can cause coughing, shortness of breath, irritation to, or burning of the eyes, nose, and throat, chest pain, wheezing, fluid in the lungs, and nausea. Chlorine can also be absorbed through the skin and cause pain, inflammation, swelling, and blistering.
* Strong oxidizer. May initiate or promote combustion in other materials.

==Occupational Exposure Limits (OELs)==
* MIOSHA: 0.5 ppm Chlorine (from sodium hypochlorite), 8-hour PEL
* MIOSHA: 1 ppm Chlorine (from sodium hypochlorite), 15-minute STEL
* AIHA (WEEL): 2 mg/m3 Sodium Hypochlorite, 15-minute STEL

==Engineering Controls==
* An eyewash/drench hose combination unit must be available in the immediate work area for any work with corrosive materials, including bleach.
* If large quantities will be used, a safety shower will also be necessary. Contact OSEH at (734) 647-1143 for a determination of the need for a safety shower if there is not one available.
* Depending on the material’s pH or based on its ability to cause severe tissue damage, e.g., formaldehyde, methylene chloride, methyl ethyl ketone peroxide (MEKP), phenol, etc., the location of the emergency shower and/or emergency eyewash shall be within 25 to 100 feet from the hazardous operation.
* A system of local and/or general exhaust is recommended to keep employee exposures below MIOSHA Permissible Exposure Limits (PEL). Local exhaust ventilation (LEV) is generally preferred because it can control the emissions of the contaminant at its source, preventing dispersion of it into the general work area. Contact OSEH at (734) 647-1143 for a determination of the need for an LEV system, if there is not one available.

==Work Practice Controls==
* ALWAYS review the SDS of both bleach and any chemicals/agents before mixing them with bleach to ensure compatibility. A general list of incompatible materials can be found here.
* NEVER mix bleach with an unknown liquid or unknown residue.
* Do not mix bleach with any compound that is incompatible with oxidizers.
* Purchase bleach in the smallest containers that are practical for lab use.
* Work with the smallest practicable amount and lowest practicable concentration.

==Personal Protective Equipment (PPE)==
In order to select the appropriate PPE for the workplace, a Hazard Assessment is conducted. The hazard assessment determines the hazards and potential hazards associated with a task, machinery, or process. The appropriate PPE for the situation may be subsequently determined. The Hazard Assessment Form may be found here. It may be completed either by the workplace supervisor or the department’s OSEH representative.
* Personal protective equipment is especially important. Wear a buttoned lab coat, safety glasses (that meet the requirements of ANSI/ISEA Z87.1) or goggles if splashing may occur, and gloves for any work with bleach. Depending on the quantities and concentrations used, a face shield, impenetrable, chemical apron and sleeves (or coverall), and special gloves may be recommended.
* Should adequate dilution ventilation or LEV be present, respiratory protection should not be necessary. However, in the absence of these and when MIOSHA PEL, or other published occupational exposures limits (OEL) are anticipated to be exceeded, respiratory protection may be necessary. Contact OSEH at (734) 647-1143 for a determination of the need for a respirator.

==Transportation and Storage==
* Transport concentrated bleach solutions in secondary containment, preferably a polyethylene or other non-reactive acid/solvent bottle carrier.
* Store in well-ventilated areas with secondary containment, such as a non-reactive plastic bin.
* Store below eye level.
* Store away from metal (unless the metal has a corrosion-proof coating), and do not store under the sink.
* Store away from incompatibles and flammable materials. Always review the SDS of other chemicals in the storage area for compatibility with bleach.
* Avoid storing on the floor. If storing on the floor is necessary, use secondary containment.

==Waste Disposal==
If the bleach solution is at a household concentration or less, i.e., a maximum 10% concentration of sodium hypochlorite, and was used for disinfecting, limit discharges down a laboratory sink to less than 1-gallon (approx. 3.8 L). If the concentration was >10% and was used for disinfecting, limit the sink disposal to less than 2-cups (approx. 500 mL) of bleach solution. Also flush the drain with adequate volumes of water.

This does not apply to “waste” bleach, e.g., unused or expired bleach. Any leftover, unused product or expired bleach not used as intended by the manufacturer, or used as a disinfectant, needs be collected by OSEH Hazardous Materials Management (HMM) for proper disposal. Contact OSEH-HMM at (734) 763-4568 for waste containers, labels, manifests, and waste collection. Also refer to the OSEH Hazardous Waste webpage for more information.

==Exposures/Unintended Contact==
If the employee is in need of emergency medical attention, call 911 immediately.

In general, flush affected eyes or skin with water for at least 15 minutes, then seek medical attention (see below).
* Eyes: In case of contact, immediately flush eyes with plenty of water for at least 15 minutes. Get medical aid.
* Skin: In case of contact, immediately flush skin with plenty of water. Remove contaminated clothing and shoes. Get medical aid. Wash clothing before reuse.
* Ingestion: Get medical aid immediately. Do not induce vomiting unless directed to do so by medical personnel. Never give anything by mouth to an unconscious person. If vomiting occurs naturally, have victim lean forward. Wash mouth with water, and then give plenty of milk or water to drink and obtain urgent medical attention.
* Inhalation: If inhaled, remove to fresh air. If not breathing, give artificial respiration. Get medical aid.

Contact OSEH for advice on symptoms of chemical exposure, or assistance in performing an exposure assessment.

Report all work related accidents, injuries, illnesses or exposures to WorkConnections within 24 hours by completing and submitting the Illness and Injury Report Form. Follow the directions on the WorkConnections website Forms Instructions to obtain proper medical treatment and follow-up.

Complete the OSEH Laboratory Incident and Near-Miss Report form.

==Treatment Facilities==
* U-M Occupational Health Services -- Campus Employees
Mon-Fri 7:30 am - 4:30 pm
After hours - go to UM Hospital Emergency Dept. – Urgent Care Clinic
C380 Med Inn building
1500 East Medical Center Drive, Ann Arbor (734) 764-8021
* University Health Services -- University students (non-life threatening conditions)
Mon-Fri 8 am – 4:30 pm, Sat 9 am – 12 pm
Contact for current hours as they may vary
207 Fletcher Street, Ann Arbor (734) 764-8320
* UMHS Emergency Department -- after clinic hours or on weekends
1500 East Medical Center Drive, Ann Arbor, (734) 936-6666

==Spill Procedures==
* When a spill occurs, personal safety should always come first.
* Alert and clear everyone in the immediate area where the spill occurred.

A minor (small) chemical spill is one that the laboratory staff is capable of handling safely without the assistance of safety and emergency personnel, i.e., less than 1 Gallon or 3.5 Liters. A major/large chemical spill requires active assistance from emergency personnel.

For minor (small) spills
* Use proper personal protective equipment as indicated above.
* Absorb the spilled bleach using an absorbent, non-combustible material such as appropriate sorbent pads, sand, or vermiculite. Do not use combustible materials such as sawdust.
* Collect residue, place in container and contact OSEH-HMM (734) 763-4568 for proper disposal.

For major (large) spills
Report large chemical spills greater than 1 gallon or 3.5 liters in corridors or common areas, e.g., hallways, elevators, eating areas, rest rooms, offices, etc., to University of Michigan Division of Public Safety and Security (DPSS) by calling 911.

Additional Spill Response Steps:

===Minor Chemical Spill===
* Alert people in immediate area of spill.
* Open outside windows, if possible.
* Wear protective equipment, including safety goggles, gloves and long-sleeve lab coat.
* Avoid breathing vapors from spill.
* Confine spill to as small an area as possible.
* Do not wash spill down the drain.
* Use appropriate spill kits/sorbents to neutralize corrosives and/or absorb spill. Collect contaminated materials and residues and place in container. For powdered chemicals sweep carefully to avoid generation of dust or, if appropriate, use moist sorbent pads or wet the powder with a suitable solvent and then wipe with a dry cloth. Contact OSEH-HMM (734) 763-4568 for proper disposal.
* Clean spill area with water.

===Major Chemical Spill===
* Attend to injured or contaminated persons and remove them from exposure.
* Alert people in the laboratory to evacuate.
* Close doors to affected area.
* Post warnings to keep people from entering the area.
* Have person available that has knowledge of incident and laboratory to assist emergency personnel.

===Additional Spill Links===
* http://www.oseh.umich.edu/pdf/chemspil.pdf
* http://www.oseh.umich.edu/emer-chemical.shtml

Report all emergencies, suspicious activity, injuries, spills, and fires to the University of Michigan Division of Public Safety and Security (DPSS) by calling 911 or texting 377911. Register with the University of Michigan Emergency Alert System via Wolverine Access.

==Training of Personnel==
All personnel are required to complete the General Laboratory Safety Training session (BLS025w or equivalent) via OSEH’s My LINC website. Furthermore, all personnel shall read and fully adhere to this SOP when handling bleach.

==Certification==
I have read and understand the above SOP. I agree to contact my Supervisor or Lab manager if I plan to modify this procedure. Sign by logging in and typing <nowiki>* ~~~~</nowiki> in the list below:

* [[User:Reddj|Reddj]] ([[User talk:Reddj|talk]]) 16:42, 12 October 2016 (UTC)
* [[User:Ccousz|Ccousz]] ([[User talk:Ccousz|talk]])
*[[User:Iharvey|Iharvey]] ([[User talk:Iharvey|talk]]) 21:17, 10 November 2016 (UTC)
* [[User:Snyderds|Snyderds]] ([[User talk:Snyderds|talk]]) 18:47, 7 June 2017 (UTC)
* [[User:Elhabbal|Elhabbal]] ([[User talk:Elhabbal|talk]]) 21:45, 12 June 2017 (UTC)
*[[User:Mollyec|Mollyec]] ([[User talk:Mollyec|talk]]) 14:52, 13 June 2017 (UTC)
Prior Approval required – Is this procedure hazardous enough to warrant prior approval from the Laboratory Director? ☐ YES ☒ NO

Laboratory Director - Dave Bridges Revision Date - 2016-07-26

SOP - Biosafety Cabinets

2017-06-13T14:51:40Z

Mollyec: /* Certification */

[[ Category: SOP ]]
[[ Category: Lab Safety]]
=Description=
This standard operating procedure outlines the use of biosafety cabinets. Review this document and supply the information required in order to make it specific to your laboratory. In accordance with this document, laboratories should use appropriate administrative controls and personal protective equipment using biosafety cabinets.

Biological Safety Cabinets, (BSCs), also known as tissue culture hoods, are designed to provide personnel, environmental and product protection when appropriate practices and procedures are followed. Class II BSCs rely on directional movement of air to provide containment. Airflow is drawn into the front grille of the cabinet, providing personnel protection. The most commonly used BSC is a Class II A2. This type of cabinet is not suitable for volatile solvents.

BSC installation, required annual certification, decontamination and maintenance must be done by certified (accredited by the National Sanitation Foundation) professionals or by a certified OSEH technician. Maintenance and certification will be done in accordance with OSEH Engineering Controls Standard of Care #1-6.

=Work Practice Controls=
==Personal Protective Equipment:==
• Appropriate personal protective equipment (PPE) must be worn. Lab coats must be buttoned. Gloves should be pulled over the wrists of lab coat, not worn inside coat. Additional PPE to be used as recommended.

==Preparing BSC for work:==
• Confirm BSC annual (within 12 months) certification is current; information found on sticker on front of BSC.

• Operate cabinet blowers at least 3-5 minutes before beginning work to allow the BSC to “purge” particulates.

• Use 70% ethanol to clean work surface of BSC and to disinfect any glass, etc. that is being used; amount of alcohol in BSC must be for only one day’s work.

==Working in the BSC:==
¥ When working in the cabinet, move arms in and out slowly, perpendicular to the face opening to reduce disruption of air curtain.

¥ Perform all operations at least 4 inches from the front grille on the work surface.

¥ For BSC clean-up, apply 70% ethanol using wipes vs. spray bottles to minimize solvent vapor concentrations being re-circulated in the hood. Cabinet sash to remain open to allow for alcohol evaporation; sash can be lowered after sufficient time. The recommended minimum time for sash opening is 10 minutes.

¥ Do not bring potentially contaminated materials out of the cabinet until they have been surface decontaminated.

¥ Disposable underpads can be placed on the work surface but must not cover the front or rear grille openings. The use of toweling facilitates routine cleanup and reduces splatter and aerosol generation during an overt spill.

¥ Place all material as far back in the cabinet as practical, toward the rear edge of the work surface and away from the front grille of the cabinet.

¥ Place aerosol-generating equipment (e.g. vortex mixers, tabletop centrifuges) toward the rear of the cabinet.

¥ The workflow should be from “clean to dirty”. Materials and supplies should be placed in the cabinet in such a way as to limit the movement of “dirty” items over “clean” ones.

==Open Flames in a BSC:==
• Open flames in BSC are not to be used.

• If a researcher requests to use open flames, OSEH personnel will meet with the researcher and discuss issues and solutions.

• If it is deemed absolutely necessary for the work being done, use a pilotless burner or safety touch-plate microburners to provide a flame on demand. Refer to OSEH Advisory Use of Bunsen Burners in Biological Safety Cabinets for guidance.

• The University of Michigan has taken a strong stance against the use of gas burners or alcohol flames in BSC. This decision has been made in accordance with recommendations from numerous agencies. The Centers for Disease Control and Prevention (CDC) reports that “open-flames are not required in the near microbe-free environment of a biological safety cabinet” and create “turbulence which disrupts the pattern of air supplied to the work surface," jeopardizing the sterility of the work area. This is also the recommendation of the World Health Organization (WHO) as well as the major BSC manufacturers.

• Early microbiologists had to rely on open flames to ensure sterility. With the advancement of modern technology, including the introduction of the BSC, the use of an open flame is no longer necessary.

==UV Lights in a BSC:==
The Center for Disease Control (CDC) and the National Institutes of Health (NIH) agree that UV lamps are not recommended nor required in BSC. UV lamps must be turned off when the room is occupied to protect eyes and skin from UV exposure, which can burn the cornea and cause skin cancer. Proper use and cleaning of BSC negates any need for the use of UV lamps. Numerous factors affect the activity of the germicidal effect of UV light, which require regular cleaning, maintenance and monitoring to ensure germicidal activity.

=Protective Equipment=
Appropriate personal protective equipment (PPE) must be worn. Lab coats must be buttoned. Gloves should be pulled over the wrists of lab coat, not worn inside coat. Additional PPE to be used as recommended.

Note: Respirators are masks designed to protect the wearer from specific airborne hazards and are different from surgical masks, which protect the wearer only from splashes and are primarily intended to protect others from infectious aerosols exhaled by the wearer. Respirator use requires employee participation in the Respiratory Protection Program, which involves medical clearance and annual fit testing and training. Please be clear about use of surgical masks versus respirators. (Do NOT use the vague term “masks”).

=Waste Disposal=
A vacuum flask system is required to provide protection to the central building vacuum system or vacuum pump and to personnel who service the equipment. The figure below illustrates a proper set-up for handling liquid waste.

--missing picture--
The left suction flask (A) is used to collect the contaminated fluids into a suitable decontamination solution; the right flask (B) serves as a fluid overflow collection vessel. An in-line HEPA filter (C) is used to protect the vacuum system (D) from microorganisms.

• Connect the primary flask to an overflow collection flask and to an in-line HEPA filter.

• Both flasks shall contain an appropriate disinfectant for the material used.

• The vacuum flasks may be set up within the cabinet; however, to save room, the system can be placed on the floor beneath or next to the BSC, using a secondary container to contain the flasks and a longer hose connection to the vacuum system.

• Once inactivation occurs, liquid materials can be disposed of as noninfectious waste in the sink. Empty the waste from the flask when it reaches no higher than ¾ full. Replace the flask with fresh disinfectant.

Questions regarding waste pick up should be directed to OSEH Hazardous Materials Management (HMM) at (734) 763-4568. This office can also assist you in minimizing waste generation, providing waste containers, labels, and manifests.

=Exposures/Unintended Contact=

If the employee is in need of emergency medical attention, call 911 immediately.

Contact OSEH for advice on symptoms of chemical exposure, or assistance in performing an exposure assessment.

Report all work related accidents, injuries, illnesses or exposures to WorkConnections within 24 hours by completing and submitting the Illness and Injury Report Form. Follow the directions on the WorkConnections website Forms Instructions to obtain proper medical treatment and follow-up.

Complete the OSEH Laboratory Incident and Near-Miss Report form.

===TREATMENT FACILITIES:===
U-M Occupational Health Services -- Campus Employees
Mon-Fri 7:30 am - 4:30 pm

After hours - go to UM Hospital Emergency Dept. – Urgent Care Clinic
C380 Med Inn building
1500 East Medical Center Drive, Ann Arbor (734) 764-8021

University Health Services -- University students (non-life threatening conditions)
Mon-Fri 8 am – 4:30 pm, Sat 9 am – 12 pm
Contact for current hours as they may vary
207 Fletcher Street, Ann Arbor (734) 764-8320

UMHS Emergency Department -- after clinic hours or on weekends
1500 East Medical Center Drive, Ann Arbor, (734) 936-6666

Click here for additional accident and injury reporting information.

=Spill Procedure=
• When a spill occurs, personal safety should always come first.

• Alert and clear everyone in the immediate area where the spill occurred.

A minor (small) biological spill is one that the laboratory staff is capable of handling safely without the assistance of safety and emergency personnel, i.e., low concentration of infectious material that has high infectious dose small volume less than 1 Liter. A major/large biological spill, i.e., high concentration of infectious material that has low infectious dose, regardless of volume outside of BSC --may require active assistance from emergency personnel if volume is significant.

==Spill Response Steps:==

===MINOR or MAJOR BIOLOCIAL SPILL in BSC===
• BSC must be operating to provide user protection.

• Alert people in immediate area of spill.

• Wear protective equipment, including safety goggles, gloves and long-sleeve lab coat.

• Decontaminate all surfaces and items before removing from BSC.

• Cover spill with paper towel then saturate covered spill with disinfectant.

• Allow disinfectant 20 minutes of contact time before wiping up spill.

• Use tongs/forceps to pick up paper towel for disposal.

• Clean spill area with fresh towels soaked in disinfectant.

• Collect all waste materials in autoclave bag and autoclave using appropriate procedures.

• Clean spill area with water or 70% ethanol to preserve integrity of BSC.

• Allow cabinet to run an additional 10 minutes before resuming work or shutting down.

===MAJOR BIOLOGICAL SPILL outside BSC===
• Attend to injured or contaminated persons and remove them from exposure.

• Alert people in the laboratory to evacuate.

• Close doors to affected area.

• Post warnings to keep people from entering the area.

• Have person available that has knowledge of agents in use, incident, and laboratory to assist emergency personnel if assistance is requested.

• Wear PPE (gloves, lab coat, eye and respiratory protection).

• Cover spill with paper towel and saturate spill with disinfectant.

• Allow disinfectant 20 minutes of contact time before wiping up spill.

• Use tongs/forceps to pick up paper towel for disposal.

• Clean spill area with fresh towels soaked in disinfectant.

• Collect all waste materials in autoclave bag and autoclave using appropriate procedures.

Additional Spill Links:
• www.oseh.umich.edu/pdf/chemspil.pdf

• http://www.oseh.umich.edu/emer-chemical.shtml.

Report all emergencies, suspicious activity, injuries, spills, and fires to the University of Michigan Division of Public Safety and Security (DPSS) by calling 911 or texting 377911. Register with the University of Michigan Emergency Alert System via Wolverine Access.

=Training of Personnel=
All personnel are required to complete the General Laboratory Safety Training session (BLS025w or equivalent) via OSEH’s My LINC website. Furthermore, all personnel shall read and fully adhere to this SOP when working within a BSC.

=Certification=
I have read and understand the above SOP. I agree to contact my Supervisor or Lab manager if I plan to modify this procedure.

*[[User:Iharvey|Iharvey]] ([[User talk:Iharvey|talk]]) 19:56, 10 November 2016 (UTC)
* [[User:Elhabbal|Elhabbal]] ([[User talk:Elhabbal|talk]]) 22:18, 12 June 2017 (UTC)
*[[User:Mollyec|Mollyec]] ([[User talk:Mollyec|talk]]) 14:51, 13 June 2017 (UTC)

Prior Approval required – Is this procedure hazardous enough to warrant prior approval from the Principal Investigator? ☐ YES ☐ NO

Principal Investigator

SOP - Needle Capping

2017-06-13T14:50:28Z

Mollyec: /* Certification */

[[ Category: SOP ]]
[[ Category: Lab Safety ]]

==Description==
This procedure outlines the methods for the safe recapping of needles and covers the safe handling, practices, disposal, and needlestick reporting requirements pertaining to hypodermic needles, hypodermic needles with syringes, needles with attached tubing, and other needle equipment. It is the responsibility of all laboratory staff to contribute to and comply with these procedures.

==Potential Hazards==
Needles present serious health hazards because people can be easily exposed parenterally to hazardous chemicals and infectious biological agents through injection. Recapping needles is extremely dangerous because it can result in the accidental stabbing of hands and other body parts. If working with human blood or other potentially infectious material, ensure that an Exposure Control Plan is completed and available. All applicable work requirements such as SOPs, universal precautions, hepatitis vaccination, etc. are required to be followed.

==Engineering Controls/Workplace Controls==
NEEDLES SHOULD NOT BE RECAPPED, BENT, REMOVED OR OTHERWISE MANIPULATED BY HAND.

* However, if it is essential that a needle be recapped due to the nature of the work, the use of a mechanical device or the one-handed scoop method must be used. In addition, there needs to be written justification and instructions for any procedures that involves the recapping of needles (SOP).

==Needle Recapping Instructions:==
1. One-Handed Scoop Method

Step 1: Place the cap on the desk or other flat surface with something firm to “push” the needle cap against.

Step 2: Holding the syringe with needle attached in one hand, slip the needle into the cap without using the other hand.

Step 3: Push the capped needle against a firm object to “seat” the cap onto the needle firmly using only one hand.

2. Needle Recapping Devices

These are devices with a hole in the center that hold needle caps and allow for single hand uncapping and recapping.

==Additional Guidelines==
* Keep needles away from fingers and other body parts.
* Place uncapped needles in a rigid tray during procedures and transportation.
* Do not leave unprotected needles on work surfaces.
* Shearing or breaking of contaminated needles is prohibited.
* Use disposable needle locking syringe units and other engineered sharps injury protections, which has a built-in safety feature or mechanism that effectively reduce exposures, and needleless systems whenever possible.
* Blunt cannulas should be used as alternatives to needles wherever possible.
* Needles and syringes should never be used as a substitute for pipettes.
* Used disposable needles and syringes must be placed in conveniently located appropriate sharps disposal containers and discarded as infectious waste.
* Use extreme caution and be alert when handling sharps. Avoid distractions and keep sight of the needle.
* Ensure that animals are restrained when using needles on animals and be on the alert for any unexpected movements.
* The use of needles and syringes should be restricted to procedures for which there is no alternative.
* Refer to the OSEH Exposure Control Plan and Biosafety Manual for additional information.

==Personal Protective Equipment (PPE)==
Wear safety glasses/goggles, gloves, lab coat/gown, close-toe shoes, and any other applicable PPE when handling needles and syringes.

==Waste Disposal==
Upon completion of a procedure involving use of needle equipment, immediately discard the items, including syringes connected to the needle, tubing, etc., into a clearly and properly labeled or color-coded rigid, closable, leakproof, puncture-resistant, disposable sharps container located in the work area at the point of use. All used needle equipment waste must be discarded as infectious waste and in compliance with OSEH requirements. NEVER reach into the sharps container. Never empty the contents into another container. Do not overfill the sharps container.

==Accidental Needle Stick/Exposures/Unintended Contact==
If the employee is in need of emergency medical attention, call 911 immediately.

If a needlestick injury should occur, wash the puncture area with soap and water, report the incident to the supervisor and principal investigator, and seek medical attention as soon as possible. Any needlestick with recombinant DNA and/or an infectious agent must be reported to the U-M Biological Safety Officer (BSO) (763-3133) as soon as possible. The U-M BSO will determine if institutional notification to Federal Agencies is required within 24 hours per applicable regulations. Failure to report may result in severe penalties to the researcher, laboratory, department or institution.
Report all needlesticks to WorkConnections within 24 hours by completing and submitting the Illness and Injury Report Form. Please include (1) the type and brand of device involved in the incident. (2) The work unit or work area where the exposure incident occurred. (3) An explanation of how the incident occurred. Follow the directions on the WorkConnections website Forms Instructions to obtain proper medical treatment and follow-up.

In addition, as required for all lab incidents, complete the OSEH Laboratory Incident and Near-Miss Report form.

TREATMENT FACILITIES:
U-M Occupational Health Services -- Campus Employees
Mon-Fri 7:30 am - 4:30 pm
After hours - go to UM Hospital Emergency Dept. – Urgent Care Clinic
C380 Med Inn building
1500 East Medical Center Drive, Ann Arbor (734) 764-8021
University Health Services -- University students (non-life threatening conditions)
Mon-Fri 8 am – 4:30 pm, Sat 9 am – 12 pm
Contact for current hours as they may vary
207 Fletcher Street, Ann Arbor (734) 764-8320
UMHS Emergency Department -- after clinic hours or on weekends
1500 East Medical Center Drive, Ann Arbor, (734) 936-6666
Click here for additional accident and injury reporting information.

Report all emergencies, suspicious activity, injuries, spills, and fires to the University of Michigan Division of Public Safety and Security (DPSS) by calling 911 or texting 377911. Register with the University of Michigan Emergency Alert System via Wolverine Access.

==Training of Personnel==
All personnel are required to complete the General Laboratory Safety Training session (BLS025w or equivalent) via OSEH’s My LINC website. Furthermore, all personnel shall read and fully adhere to this SOP when handling needles.

==Certification==
I have read and understand the above SOP. I agree to contact my Supervisor or Lab Manager if I plan to modify this procedure. Sign by logging in and typing <nowiki>* ~~~~</nowiki> in the list below:

* [[User:Ccousz|Ccousz]] ([[User talk:Ccousz|talk]])
*[[User:Iharvey|Iharvey]] ([[User talk:Iharvey|talk]]) 21:24, 10 November 2016 (UTC)
* [[User:Snyderds|Snyderds]] ([[User talk:Snyderds|talk]]) 19:33, 7 June 2017 (UTC)
*[[User:Reddj|Reddj]] ([[User talk:Reddj|talk]]) 14:03, 9 June 2017 (UTC)
* [[User:Elhabbal|Elhabbal]] ([[User talk:Elhabbal|talk]]) 22:00, 12 June 2017 (UTC)
*[[User:Mollyec|Mollyec]] ([[User talk:Mollyec|talk]]) 14:50, 13 June 2017 (UTC)
Prior Approval required – Is this procedure hazardous enough to warrant prior approval from the Laboratory Director? ☐ YES ☒ NO

Laboratory Director - Dave Bridges Revision Date - 2016-10-14

SOP - Centrifuge Safety

2017-06-13T14:50:04Z

Mollyec: /* Certification */

[[ Category: SOP ]]
[[ Category: Lab Safety ]]

==Description==
This standard operating procedure outlines the use of centrifuges. Review this document and supply the information required in order to make it specific to your laboratory. In accordance with this document, laboratories should use appropriate controls and personal protective equipment when using centrifuges.

Centrifuges are machines used to separate solids from liquids in a suspension. The spinning motion of a centrifuge produces centrifugal forces that separate substances of greater and lesser densities.

==Potential Hazards==
The centrifuge must be loaded carefully balanced. An unbalanced load may present a risk to both the machine and to persons nearby. Centrifuge rotors should never be touched while the rotor is spinning, as they present a significant personal risk when in motion. Another potential hazard is the possible aerosolization of harmful samples during centrifuge operation.

==Engineering Controls==
If centrifuging hazardous materials, care should be taken to use tightly capped tubes and/or sealable safety cups or rotors that can be loaded and unloaded in a fume hood or biosafety cabinet, depending on the hazard.

==Work Practice Controls==
The following information may be integrated into a lab-specific standard operating procedure (SOP) for centrifuge use.

1. ''Preventive Maintenance''

A. Establish preventive maintenance schedule: Including regular cleaning of centrifuge interior to prevent damage and avoid costly repairs. Reference centrifuge operator’s manual or contact manufacturer for guidance.

Equipment repair and adjustments shall only be conducted by qualified service technicians.

B. Maintain log book: For all high speed centrifuges and ultracentrifuges include run dates, durations, speeds, total rotor revolutions, and notes on rotor condition.

Retire rotors after manufacturer’s recommended life span except where annual stress test demonstrates absence of structural flaws. Note: Rotor life span may be reduced or warranty voided if autoclaved; contact manufacturer for guidance.

2. ''Planning for Use''

A. Complete lab-specific training for the centrifuge.

B. Wear appropriate PPE: Including safety eyewear, gloves, lab coat, and appropriate street clothing (i.e., long pants and closed-toe shoes). Ensure gloves are compatible with hazard(s).

C. Inspect centrifuge:
* Ensure tubes are rated for intended use (speed, temperature, and chemical resistance).
* Ensure rotor is compatible with centrifuge and seated on the drive correctly.
* Ensure rotor and safety cups/buckets are free of cracks and deformities.
* Ensure rotor O-ring is not cracked, missing, or worn.
* Ensure safety cups/buckets are attached correctly and able to move freely.

Contact a qualified service technician if inspection identifies centrifuge components requiring repair or replacement.

D. Prepare centrifuge tubes for loading:
* Inspect centrifuge tubes before use.
* Follow manufacturer’s filling limits for tubes. Do not overfill or underfill tubes.
* For biohazardous materials, disinfect outside of tubes prior to removal from biosafety cabinet and loading into rotor.

When centrifuging hazardous materials, use tightly capped tubes and/or sealable safety cups or rotors that can be loaded and unloaded in a fume hood or biosafety cabinet, depending on hazard. If a specific procedure does not allow for this, contact Occupational Safety and Environmental Health (OSEH) at (734) 647-1143 for guidance.

E. Use in-line filter: For high speed centrifuges and ultracentrifuges, use in-line filters to prevent contamination of vacuum pump and pump oil. Provide secondary containment for vacuum pump.

3. ''Centrifuge Operation''

A. Balance centrifuge: Follow manufacturer’s instructions for proper centrifuge balancing steps.

B. Start run
* Do not leave centrifuge until full operating speed is reached and it appears to be running safely without incident.
* Stop centrifuge immediately if you notice any unusual noises or shaking. Confirm rotor is balanced.

To prevent rotor failure, do not exceed maximum speed and maximum mass limits for the rotor. You must reduce rotor speed if sample density calculations indicate maximum mass limits will be exceeded; contact manufacturer for guidance.

4 ''Sample Removal''

A. Stop run: Ensure centrifuge comes to complete stop before opening cover. When centrifuging hazardous materials, wait at least 10 minutes after run to allow aerosols to settle before opening centrifuge.

B. Check for leaks/spills: In samples, rotor, safety cups/buckets, and centrifuge well.

C. Open sealable tubes/safety cups/rotors: Wear appropriate PPE and open inside fume hood or biosafety cabinet, depending on hazard.

==Personal Protective Equipment==
When operating the centrifuge, proper eye protection (glasses/goggles), gloves, lab coat, and close-toe footwear should be worn. Gloves should be appropriate for the present hazard.

==Waste Disposal==
Centrifuge Disposal

1. For biohazardous materials, clean/disinfect centrifuge and remove/cross out biohazard sticker. Attach note on centrifuge describing what has been done.

2. For radioactive materials, request radiation safety survey and signage before disposal of centrifuge.

==Exposures/Unintended Contact==

If the employee is in need of emergency medical attention, call 911 immediately.

Contact OSEH for advice on symptoms of chemical exposure, or assistance in performing an exposure assessment.

Report all work related accidents, injuries, illnesses or exposures to WorkConnections within 24 hours by completing and submitting the Illness and Injury Report Form. Follow the directions on the WorkConnections website Forms Instructions to obtain proper medical treatment and follow-up.

Complete the OSEH Laboratory Incident and Near-Miss Report form.

TREATMENT FACILITIES:
U-M Occupational Health Services -- Campus Employees
Mon-Fri 7:30 am - 4:30 pm
After hours - go to UM Hospital Emergency Dept. – Urgent Care Clinic
C380 Med Inn building
1500 East Medical Center Drive, Ann Arbor (734) 764-8021
University Health Services -- University students (non-life threatening conditions)
Mon-Fri 8 am – 4:30 pm, Sat 9 am – 12 pm
Contact for current hours as they may vary
207 Fletcher Street, Ann Arbor (734) 764-8320
UMHS Emergency Department -- after clinic hours or on weekends
1500 East Medical Center Drive, Ann Arbor, (734) 936-6666
Click here for more information.

==Spill Procedure==
1. Mechanical Failure

Turn off centrifuge immediately and unplug power cord. Do not use centrifuge again until inspected by qualified service technician.

2. Hazardous Material Spill/Exposure

A. Turn off centrifuge immediately. Keep centrifuge cover closed for at least 30 minutes to reduce aerosolization of hazardous material.

B. Follow spill, exposure, and incident reporting instructions.
* For chemical, radioactive, and biohazard spills, contact OSEH at (734) 647-1143 for assistance and guidance.

Remember:
* Conduct rotor or safety cup/bucket cleanup in nearest biosafety cabinet or fume hood, depending on hazard.
* Use tongs or forceps to avoid contact with sharp-edged debris.
* Avoid alkaline cleaners for aluminum centrifuge components.
* Avoid abrasive wire brushes for cleaning.

Report all emergencies, suspicious activity, injuries, spills, and fires to the University of Michigan Division of Public Safety and Security (DPSS) by calling 911 or texting 377911. Register with the University of Michigan Emergency Alert System via Wolverine Access.

==Training of Personnel==
All personnel are required to complete the General Laboratory Safety Training session (BLS025w or equivalent) via OSEH’s My LINC website. Furthermore, all personnel shall read and fully adhere to this SOP when handling centrifuges.

==Certification==
I have read and understand the above SOP. I agree to contact my Supervisor or Lab manager if I plan to modify this procedure. Sign by logging in and typing <nowiki>* ~~~~</nowiki> in the list below:

* [[User:Ccousz|Ccousz]] ([[User talk:Ccousz|talk]]) 17:19, 14 October 2016 (UTC)
*[[User:Iharvey|Iharvey]] ([[User talk:Iharvey|talk]]) 21:18, 10 November 2016 (UTC)
*[[User:Pfeiferl|Pfeiferl]] ([[User talk:Pfeiferl|talk]]) 18:09, 5 June 2017 (UTC)
* [[User:Snyderds|Snyderds]] ([[User talk:Snyderds|talk]]) 18:49, 7 June 2017 (UTC)
*[[User:Reddj|Reddj]] ([[User talk:Reddj|talk]]) 14:01, 9 June 2017 (UTC)
* [[User:Elhabbal|Elhabbal]] ([[User talk:Elhabbal|talk]]) 21:59, 12 June 2017 (UTC)
*[[User:Mollyec|Mollyec]] ([[User talk:Mollyec|talk]]) 14:50, 13 June 2017 (UTC)

Prior Approval required – Is this procedure hazardous enough to warrant prior approval from the Laboratory Director? ☐ YES ☒ NO

Laboratory Director - Dave Bridges Revision Date - 2016-10-14

SOP - Formalin and Paraformaldehyde

2017-06-13T14:49:31Z

Mollyec: /* Certification */

[[ Category: SOP ]]
[[ Category: Lab Safety ]]

'''Laboratory Director (LD) Approval is Required Prior to Performing this Procedure'''

==Description==
This standard operating procedure outlines the handling and use of formalin and paraformaldehyde. Review this document and supply the information required in order to make it specific to your laboratory. In accordance with this document, laboratories should use appropriate controls, personal protective equipment, and disposal techniques when handling formalin and paraformaldehyde.

Formalin and paraformaldehyde are primarily used as fixatives. These fixatives act to preserve and stabilize cells and tissues prior to examination processes. The aqueous solution of formalin is 37-40 percent formaldehyde in water or methanol. Paraformaldehyde is the crystallized polymer of formaldehyde (97%) that is weighed out and dissolved in solution for experimentation or for cell and tissue fixation. Typically 3-10% formalin or paraformaldehyde solutions are used to perfuse or fix tissues.

==Useful Formalin and Paraformaldehyde Links:==
http://www.cdc.gov/niosh/docs/81-111/

http://www.cdc.gov/niosh/topics/formaldehyde/

http://www.cdc.gov/niosh/npg/npgd0293.html

==Potential Hazards==
*Formalin and paraformaldehyde solutions can emit formaldehyde gas, a known human carcinogen, and can irritate the eyes and skin.
*Working with paraformaldehyde powder (and, to a lesser extent, flakes or granules), can expose employees to paraformaldehyde dust, which is a strong irritant/sensitizer.
*Contact with these solutions or paraformaldehyde solids may also cause drying of the skin and/or allergic dermatitis.
*The MIOSHA Permissible Exposure Limit for formaldehyde is 0.75 ppm for 8 hours or 2 ppm for 15 minutes. There is a substance-specific MIOSHA standard for formaldehyde, and an action limit of 0.5 ppm.
*Consult your Safety Data Sheet for more information on hazards.

==Engineering Controls==
*Work with concentrated (>4% formaldehyde/paraformaldehyde) solutions only in a chemical fume hood.
*Handle paraformaldehyde powder (and, preferably, granules or flakes) only in a chemical fume hood.
*Dilute solutions (<4% formaldehyde) may be used on the benchtop in small quantities.
*If there is any possibility that an employee's eyes may be splashed with solutions containing 0.1 percent or greater formaldehyde, an eyewash/drench hose must be available within the immediate work area for emergency use.
*If employees' skin may become splashed with solutions containing 1 percent or greater formaldehyde, for example, because of equipment failure or improper work practices, the MIOSHA formaldehyde standard requires a conveniently-located safety shower. Contact Occupational Safety and Environmental Health (OSEH) at (734) 647-1143 to determine if a safety shower will be needed.

==Work Practice Controls==
*Designate an area for working with concentrated formalin, concentrated paraformaldehyde solutions, and paraformaldehyde solid, and label it as such.
*Keep containers closed as much as possible.
*Use in the smallest practical quantities for the experiment being performed.
*If you are weighing paraformaldehyde powder and the balance cannot be located in a fume hood or BSC, tare a container then add powder in the hood and cover before returning to the balance to weigh the powder.
*Labs handling moderate to large quantities of formaldehyde-containing solutions on a regular basis should contact OSEH at (734) 647-1143 for assessment of exposure. Areas that handle only small (100 ml or less) pre-filled specimen containers, or that work with formaldehyde-containing solutions exclusively in a functioning chemical fume hood, would have low potential for overexposure, but should contact OSEH if there are concerns.
*Once work with formalin/paraformaldehyde is complete, wipe down area with a soap and water solution.

==Protective Equipment==
Wear standard nitrile laboratory gloves, chemical splash goggles, face shield, and lab coat. If splash may occur, also wear an impervious apron. (MIOSHA requires that all contact of the eyes and skin with liquids containing 1 percent or more formaldehyde be prevented by the use of chemical protective clothing made of material impervious to formaldehyde and the use of other personal protective equipment, such as goggles and face shields, as appropriate to the operation.)

==Transportation and Storage==
*Transport formaldehyde solutions in secondary containment, preferably a polyethylene or other non-reactive acid/solvent bottle carrier.
*Keep container in cool, well-ventilated area.
*Keep container tightly closed and sealed until ready for use.
*Store in secondary containment with flammables, away from oxidizers, reducing agents, metals, and acids.
*Keep containers of paraformaldehyde (PFA) solid away from water.
*Avoid storing on the floor.
*Avoid ignition sources.

==Waste Disposal==
Formalin and paraformaldehyde solutions and powders must be disposed following the guidelines above while accumulating wastes and awaiting chemical waste pickup. Waste must be disposed of following OSEH Hazardous Materials procedures. Contact OSEH’s Hazardous Materials Management (HMM) at (734) 763-4568 for waste containers, labels, manifests, and waste collection. Also refer to OSEH’s Hazardous Waste webpage for more information.

==Exposures/Unintended Contact==

'''If the employee is in need of emergency medical attention, call 911 immediately.
'''
For an actual chemical exposure/injury,
*Flush exposed eyes or skin with water for at least 15 minutes, then seek medical attention (see below).
*If there is respiratory irritation associated with exposure, remove all persons from the contaminated area.

Contact OSEH for advice on symptoms of chemical exposure, or assistance in performing an exposure assessment.

Report all work related accidents, injuries, illnesses or exposures to WorkConnections within 24 hours by completing and submitting the Illness and Injury Report Form. Follow the directions on the WorkConnections website Forms Instructions to obtain proper medical treatment and follow-up.

Complete the OSEH Laboratory Incident and Near-Miss Report form.

==Treatment Facilities:==
*U-M Occupational Health Services -- Campus Employees
Mon-Fri 7:30 am - 4:30 pm
After hours - go to UM Hospital Emergency Dept. – Urgent Care Clinic
C380 Med Inn building
1500 East Medical Center Drive, Ann Arbor (734) 764-8021
*University Health Services -- University students (non-life threatening conditions)
Mon-Fri 8 am – 4:30 pm, Sat 9 am – 12 pm
Contact for current hours as they may vary
207 Fletcher Street, Ann Arbor (734) 764-8320
*UMHS Emergency Department -- after clinic hours or on weekends
1500 East Medical Center Drive, Ann Arbor, (734) 936-6666

Click here for more information.

==Spill Procedure==
Employees in the area should be prepared to clean up minor spills, including most spills confined to the chemical fume hood. Wearing double nitrile gloves, splash goggles, face shield and lab coat (and impermeable apron, if available), use absorbent pads to absorb spilled material. For small spills of solid PFA, dampen the absorbent pad with methanol before placing over the spilled material and allow to sit for a few minutes before wiping up. After spill has been completely absorbed, wash down contaminated area with soap and water at least two times. Contaminated PPE and clean-up materials must be placed in a clear plastic bag or compatible container for pick-up by OSEH.

==Additional Spill Links:==
*www.oseh.umich.edu/pdf/chemspil.pdf
*http://www.oseh.umich.edu/emer-chemical.shtml.

For large spills and most spills of formalin or paraformaldehyde solutions or paraformaldehyde powder that occurs outside of a chemical fume hood should be referred to OSEH-HMM.

'''Report all emergencies, suspicious activity, injuries, spills, and fires to the University of Michigan Division of Public Safety and Security (DPSS) by calling 911 or texting 377911. Register with the University of Michigan Emergency Alert System via Wolverine Access.
'''

==Training of Personnel==
All personnel are required to complete the General Laboratory Safety Training session (BLS025w or equivalent) via OSEH’s My LINC website. Furthermore, all personnel shall read and fully adhere to this SOP when handling formalin and paraformaldehyde.

==Certification==
I have read and understand the above SOP. I agree to contact my Supervisor or Lab manager if I plan to modify this procedure. Sign by logging in and typing <nowiki>* ~~~~</nowiki> in the list below:

*[[User:Reddj|Reddj]] ([[User talk:Reddj|talk]]) 15:58, 13 October 2016 (UTC)
*[[User:Iharvey|Iharvey]] ([[User talk:Iharvey|talk]]) 21:23, 10 November 2016 (UTC)
* [[User:Snyderds|Snyderds]] ([[User talk:Snyderds|talk]]) 19:31, 7 June 2017 (UTC)
* [[User:Elhabbal|Elhabbal]] ([[User talk:Elhabbal|talk]]) 21:50, 12 June 2017 (UTC)
*[[User:Mollyec|Mollyec]] ([[User talk:Mollyec|talk]]) 14:49, 13 June 2017 (UTC)
Laboratory Director - Dave Bridges Revision Date - 2016-10-13

SOP - Carcinogens

2017-06-13T14:48:49Z

Mollyec: /* Certification */

[[ Category: SOP ]]
[[ Category: Lab Safety ]]

'''Laboratory Director (LD) Approval is Required Prior to Performing this Procedure
'''
==Description==
This standard operating procedure is designed to provide guidance in writing procedures for the safe handling and disposal of carcinogens, which are regarded as particularly hazardous substances. Review this document and supply the information required in order to make it specific to your laboratory.

A carcinogen is defined by meeting one the following:
*It is listed under the category, “known to be carcinogens” in the latest National Toxicology Program annual report.
*It is listed under Group 1 by the International Agency for Research on Cancer (IARC).
*It is listed in Group 2A or 2B by IARC or under the category, “reasonably anticipated to be carcinogens” by NTP, and causes statistically significant tumor incidence in experimental animals in accordance with any of the following criteria:
**After inhalation exposure of 6-7 hours per day, 5 days per week, for a significant portion of a lifetime to dosages of less than 10 mg/m3.
**After repeated skin application of less than 300 mg/kg of body weight per week.
**After oral dosages of less than 50 mg/kg of body weight per day.
If you have questions concerning the applicability of any item listed in this procedure contact the LD/Laboratory Supervisor or Occupational Safety and Environmental Health at (734) 647-1143.

==Potential Hazards==
A carcinogen commonly describes any agent that can initiate or speed the development of malignant or potentially malignant tumors, malignant neoplastic proliferation of cells, or cells that possess such material.

==Engineering Controls==
*Manipulation of carcinogens should be carried out in a fume hood. If the use of a fume hood proves impractical refer to the section on special ventilation.
*Certain carcinogens must be handled in a glove box rather than a fume hood. Occupational Safety and Environmental Health (734) 647-1143 or the LD will determine if this is required.
*Where the eyes or body of any person may be exposed to carcinogens, suitable facilities for quick drenching or flushing of the eyes and body shall be provided within the work area for immediate emergency use. Bottle type eyewash stations are not acceptable.
*Safety shielding is required any time there is a risk of explosion, splash hazard or a highly exothermic reaction. All manipulations of carcinogens which pose this risk should occur in a fume hood with the sash in the lowest feasible position. Portable shields, which provide protection to all laboratory occupants, are acceptable.
*Manipulation of carcinogens outside of a fume hood may require special ventilation controls in order to minimize exposure to the material. Fume hoods provide the best protection against exposure to carcinogens in the laboratory and are the preferred ventilation control device. When possible, handle carcinogens in a fume hood. If the use of a fume hood proves impractical, attempt to work in a glove box or on an isolated area on the bench top.
*If available, consider using a Biological Safety Cabinet. The biological safety cabinet is designed to remove particulates (the carcinogen) before the air is discharged into the environment. Carcinogens that are volatile must not be used in a biological safety cabinet unless the cabinet is vented to the outdoors.
*If your research does not permit the handling of carcinogens in a fume hood, biological safety cabinet, or glove box, you must contact Occupational Safety and Environmental Health (OSEH).
*Evacuated glassware can implode and eject flying glass, and splattered chemicals. Vacuum work involving carcinogens must be conducted in a fume hood, glove box or isolated in an acceptable manner.
*Mechanical vacuum pumps must be protected using cold traps and, where appropriate, filtered to prevent particulate release. The exhaust for the pumps must be vented into an exhaust hood.

==Work Practice Controls==
All areas where carcinogens are stored or manipulated must be labeled as a designated area.
*The room sign for the laboratory must contain a “Designated Area Within” identifier.
*All locations within the laboratory where carcinogens are handled should be demarcated with designated area caution tape (available from OSEH, or your department) and/or posted with designated area caution signs. This includes all fume hoods and bench tops where the carcinogens are handled.
*All employees with access to designated areas must be made aware of the substances being used and necessary precautions by the LD.
*Where feasible, carcinogens should be manipulated over plastic-backed disposable paper work surfaces. These disposable work surfaces minimize work area contamination and simplify clean up.

'''Doorways:''' The room sign must contain a “Designated Area Within, Caution” where carcinogens, reproductive hazards, and/or acutely toxic chemicals are stored or used.

'''Containers:''' All containers of carcinogens must be clearly labeled with the correct chemical name. Handwritten labels are acceptable; chemical formulas and structural formulas are not acceptable.

Wash hands, forearms, face, and neck upon exit of a designated area to decontaminate.

Keep accurate records of amounts of carcinogenic substances being stored and used, including dates of use and names of researchers using the substances.

==Personal Protective Equipment==
Eye protection in the form of safety glasses must be worn at all times when handling carcinogens. Ordinary (street) prescription glasses do not provide adequate protection. (Contrary to popular opinion these glasses cannot pass the rigorous test for industrial safety glasses.) Adequate safety glasses must meet the requirements of the Practice for Occupational and Educational Eye and Face Protection (ANSI Z.87. 1) and must be equipped with side shields. Safety glasses with side shields do not provide adequate protection from splashes; therefore, when the potential for splash hazard exists, other eye protection and/or face protection must be worn.
Gloves must be worn when handling carcinogens. Disposable nitrile gloves provide adequate protection against accidental hand contact with small quantities of most laboratory chemicals. Lab workers should contact OSEH for advice on chemical resistant glove selection when direct or prolonged contact with hazardous chemicals is anticipated.
Lab coats, closed toed shoes and long sleeved clothing must be worn when handling carcinogens. Additional protective clothing should be worn if the possibility of skin contact is likely.

==Waste Disposal==
Because most spent, unused and expired chemicals/materials are considered hazardous wastes, they must be properly disposed of. Do not dispose of chemical wastes by dumping them down a sink, flushing in a toilet or discarding in regular trash containers, unless authorized by OSEH Hazardous Materials Management (HMM). Contact OSEH-HMM at (734) 763-4568 for waste containers, labels, manifests, waste collection and for any questions regarding proper waste disposal. Also refer to OSEH’s Hazardous Waste webpage for more information.

==Exposures/Unintended Contact==

'''If the employee is in need of emergency medical attention, call 911 immediately.
'''

For an actual chemical exposure/injury:
*Remove contaminated clothing. Flush exposed eyes or skin with water for at least 15 minutes. Seek medical attention (see below).
*For situations with risk of inhalation exposure (including spills of powder outside of a chemical fume hood), remove all persons from the contaminated area.
*If an ambulance is needed, call the University of Michigan Division of Public Safety and Security (DPSS) by calling 911 to request assistance.

Contact OSEH for advice on symptoms of chemical exposure, or assistance in performing an exposure assessment.

Report all work related accidents, injuries, illnesses or exposures to WorkConnections within 24 hours by completing and submitting the Illness and Injury Report Form. Follow the directions on the WorkConnections website Forms Instructions to obtain proper medical treatment and follow-up.

Complete the OSEH Laboratory Incident and Near-Miss Report form.

==Treatment Facilities:==
*U-M Occupational Health Services -- Campus Employees
Mon-Fri 7:30 am - 4:30 pm
After hours - go to UM Hospital Emergency Dept. – Urgent Care Clinic
C380 Med Inn building
1500 East Medical Center Drive, Ann Arbor (734) 764-8021
*University Health Services -- University students (non-life threatening conditions)
Mon-Fri 8 am – 4:30 pm, Sat 9 am – 12 pm
Contact for current hours as they may vary
207 Fletcher Street, Ann Arbor (734) 764-8320
*UMHS Emergency Department -- after clinic hours or on weekends
1500 East Medical Center Drive, Ann Arbor, (734) 936-6666

Click here for more information.

==Spill Procedures==
*When a spill occurs, personal safety should always come first.
*Alert and clear everyone in the immediate area where the spill occurred.

A minor (small) chemical spill is one that the laboratory staff is capable of handling safely without the assistance of safety and emergency personnel, i.e., less than 1 liter. A major/large chemical spill requires active assistance from emergency personnel.

==Additional Spill Response Steps:==

'''MINOR CHEMICAL SPILL'''
*Alert people in immediate area of spill.
*If spilled material is flammable, turn off ignition and heat sources. Don’t light Bunsen burners or turn on other switches.
*Open outside windows, if possible.
*Wear protective equipment, including safety goggles, gloves and long-sleeve lab coat.
*Avoid breathing vapors from spill.
*Confine spill to as small an area as possible.
*Do not wash spill down the drain.
*Use appropriate spill kits/sorbents to neutralize corrosives and/or absorb spill. Collect contaminated materials and residues and place in container. For powdered chemicals sweep carefully to avoid generation of dust or, if appropriate, use moist sorbent pads or wet the powder with a suitable solvent and then wipe with a dry cloth. Contact OSEH-HMM (734) 763-4568 for proper disposal.
*Clean spill area with water.

'''MAJOR CHEMICAL SPILL'''
*Attend to injured or contaminated persons and remove them from exposure.
*Alert people in the laboratory to evacuate.
*If spilled material is flammable, turn off ignition and heat sources. Don’t light Bunsen burners or turn on other switches.
*Call University of Michigan Division of Public Safety and Security (DPSS) at 911 immediately for assistance.
*Close doors to affected area.
*Post warnings to keep people from entering the area.
*Have person available that has knowledge of incident and laboratory to assist emergency personnel.

'''Additional Spill Links:'''
*www.oseh.umich.edu/pdf/chemspil.pdf
*http://www.oseh.umich.edu/emer-chemical.shtml.

Report all emergencies, suspicious activity, injuries, spills, and fires to the University of Michigan Division of Public Safety and Security (DPSS) by calling 911 or texting 377911. Register with the University of Michigan Emergency Alert System via Wolverine Access.

==Training of Personnel==
All personnel are required to complete the General Laboratory Safety Training session (BLS025w or equivalent) via OSEH’s My LINC website. Furthermore, all personnel shall read and fully adhere to this SOP when handling carcinogens.

==Certification==
I have read and understand the above SOP. I agree to contact my Supervisor or Lab manager if I plan to modify this procedure. Sign by logging in and typing <nowiki>* ~~~~</nowiki> in the list below:

*[[User:Reddj|Reddj]] ([[User talk:Reddj|talk]]) 13:32, 13 October 2016 (UTC)
*[[User:Iharvey|Iharvey]] ([[User talk:Iharvey|talk]]) 21:18, 10 November 2016 (UTC)
* [[User:Snyderds|Snyderds]] ([[User talk:Snyderds|talk]]) 18:48, 7 June 2017 (UTC)
* [[User:Elhabbal|Elhabbal]] ([[User talk:Elhabbal|talk]]) 21:51, 12 June 2017 (UTC)
*[[User:Mollyec|Mollyec]] ([[User talk:Mollyec|talk]]) 14:48, 13 June 2017 (UTC)
Laboratory Director - Dave Bridges Revision Date - 2016-10-13

SOP - Animal Anesthetics

2017-06-13T14:48:16Z

Mollyec: /* Certification */

[[ Category: SOP ]]
[[ Category: Lab Safety]]

==Description==
This standard operating procedure outlines the handling and use of animal anesthetics including: isoflurane, halothane, enflurane and ether. Review this document and supply the information required in order to make it specific to your laboratory. In accordance with this document, laboratories should use appropriate controls and personal protective equipment when handling animal anesthetics.

==Procedure Location==
The use of animal anesthetics must be performed in an area with good ventilation and controls to capture and exhaust waste anesthetic gases.

==Potential Hazards==
Anesthetic gas and vapor that leaks during medical or research procedures are considered waste anesthetic gases (WAGs). University faculty, staff and students should be aware of the potential risks of WAGs and be advised to take appropriate precautions to reduce exposures. Workers acutely exposed to excessive amounts of anesthetic gas can experience symptoms of drowsiness, headache, nausea, poor judgment and loss of coordination. Chronic symptoms of over-exposure can include liver, kidney and reproductive effects. Safety precautions include the use of an approved gas scavenging system, or using the agent inside a certified chemical fume hood.

The use of ether is not recommended because it is flammable and a mutagen. Be certain that there are no ignition sources present when handling ether. There are restrictions concerning the use of ether with animals. Contact OSEH at (734) 647-1143 concerning the use of ether.

==Engineering Controls==
Anesthetics should not be handled on the bench top without special ventilation or a scavenging system. Anesthetic gas filtering cartridges, snorkel exhaust, fume hoods or other scavenging systems must be used. ULAM provides ventilated procedure rooms designed for use of anesthetic gases in many areas. Fume hoods provide the best protection against exposure to anesthetics in the laboratory and are the preferred ventilation control device when handling greater than 100 cc outside of the original container. Always handle large quantities of ethyl ether in a fume hood due to the flammable nature of the material. If your research does not permit the handling of large quantities of ethyl ether in your fume hood, contact OSEH to review the adequacy of all special ventilation.

Liquid anesthetics administered with a vaporizer must be scavenged. When used properly, vaporizers equipped with activated charcoal canisters (e.g. F/Air) are effective in removing halogenated waste gases. The F/Air canister containing activated charcoal will absorb waste anesthetics for about 12 hours. Note: F/Air Canisters only absorb halogenated anesthetics (e.g. isoflurane, halothane). Immediately before using any anesthesia machine, the F/Air canister should be removed and weighed to evaluate the remaining absorption capacity. The weight should be recorded and dated on the side of the canister. Immediately following the use of an anesthesia machine, the number of hours the machine was in use should be recorded next to the dated weight information.

Canisters that exceed 12 hours of use or 50 grams of accumulated weight (whichever comes first) must be removed and placed in a sealed plastic bag and disposed of as a hazardous waste through OSEH Hazardous Materials Management (HMM) at (734) 763-4568. Thoroughly clean the induction chamber immediately after each use to avoid residual anesthetic waste release into the environment (which can continue to be released for up to three hours). Please refer to the OSEH Guideline entitled Anesthetic Gas Use for additional information on the safe use of anesthetic gases.

==Work Practice Controls==
All anesthetic agents must be clearly labeled with the correct chemical name. Handwritten labels are acceptable; chemical formulas and structural formulas are not acceptable.

Always keep the flow rate of anesthetics to the animal as low as possible during the procedure. High flow rates can increase your exposure to the anesthetic. It is also important to move the point of potential gas release as close to the exhaust system as possible to increase capture of the chemical.

Do not permit containers to remain open on the bench top. The odor thresholds for most liquid anesthetics (except for ether) are well above permissible exposure limits. If you smell the anesthetic the control procedures you are using are inadequate and must be re-evaluated.

==Personal Protective Equipment (PPE)==
Eye protection in the form of safety glasses must be worn at all times when handling anesthetic agents. Ordinary (street) prescription glasses do not provide adequate protection

Single use nitrile or latex gloves must be worn when handling anesthetic agents as well as lab coats, closed toed shoes and pants. Additional protective clothing should be worn if the possibility of skin contact is likely.

==Transportation and Storage==
Ethers form potentially explosive peroxides after exposure to air and light. Since these chemicals are packaged in an air atmosphere, peroxides can form even though the containers have not been opened. Write the date received and date opened on all containers of ether. Opened containers of ether should be discarded within 12 months of opening. Even closed containers of ether must be discarded by the expiration date through OSEH-HMM (734) 763-4568.

Halogenated liquid anesthetic agents (i.e. halothane, enflurane, isoflurane) are not flammable but do have limited shelf life. Be certain to date the chemical when it is opened and to check expiration date before use.

Always purchase the smallest quantity required for use. Ether used for anesthetic purposes should be purchased in the smallest quantity available (typically 150 cc, Fisher Scientific E136-150) due to its short (12 month) shelf life after it is opened.

==Waste Disposal==
Anesthetic agents are hazardous wastes. Contact OSEH-HMM at (734) 763-4568 for waste containers, labels, manifests, waste collection and for any questions regarding proper waste disposal. Also refer to OSEH’s Hazardous Waste webpage for more information.

==Exposures/Unintended Contact==

'''If the employee is in need of emergency medical attention, call 911 immediately.'''

Wash hands and arms with soap and water immediately following any skin contact with anesthetic agents. Flush eyes for 15 minutes following eye contact.

Contact OSEH for advice on symptoms of chemical exposure, or assistance in performing an exposure assessment.

Report all work related accidents, injuries, illnesses or exposures to WorkConnections within 24 hours by completing and submitting the Illness and Injury Report Form. Follow the directions on the WorkConnections website Forms Instructions to obtain proper medical treatment and follow-up.

Complete the OSEH Laboratory Incident and Near-Miss Report form.

==Treatment Facilities:==
*U-M Occupational Health Services -- Campus Employees
Mon-Fri 7:30 am - 4:30 pm
After hours - go to UM Hospital Emergency Dept. – Urgent Care Clinic
C380 Med Inn building
1500 East Medical Center Drive, Ann Arbor (734) 764-8021
*University Health Services -- University students (non-life threatening conditions)
Mon-Fri 8 am – 4:30 pm, Sat 9 am – 12 pm
Contact for current hours as they may vary
207 Fletcher Street, Ann Arbor (734) 764-8320
*UMHS Emergency Department -- after clinic hours or on weekends
1500 East Medical Center Drive, Ann Arbor, (734) 936-6666

Click here for additional accident and injury reporting information.

==Spill Procedure==
Ether is extremely flammable. If ether is spilled immediately assess and deactivate potential ignition sources. Be prepared for a potential fire and ensure your safety and others first.

Anticipate spills by having the appropriate clean up equipment on hand. Spill materials for anesthetic agents are designed to control the liquid portion of the spill and minimize the production of vapors. Never use paper towels on large spills of anesthetic agents because it exacerbates vapor production.

*When a spill occurs, personal safety should always come first.
*Alert and clear everyone in the immediate area where the spill occurred.

A minor (small) chemical spill is one that the laboratory staff is capable of handling safely without the assistance of safety and emergency personnel, i.e., less than 1 liter. A major/large chemical spill requires active assistance from emergency personnel.

==Additional Spill Response Steps:==

===MINOR CHEMICAL SPILL===
*Alert people in immediate area of spill.
*If spilled material is flammable, turn off ignition and heat sources. Don’t light Bunsen burners or turn on other switches.
*Open outside windows, if possible.
*Wear protective equipment, including safety goggles, gloves and long-sleeve lab coat.
*Avoid breathing vapors from spill.
*Confine spill to as small an area as possible.
*Do not wash spill down the drain.
*Use appropriate spill kits/sorbents to absorb spill. Collect contaminated materials and residues and place in container. Contact OSEH-HMM (734) 763-4568 for proper disposal.
*Clean spill area with water.

===MAJOR CHEMICAL SPILL===
*Attend to injured or contaminated persons and remove them from exposure.
*Alert people in the laboratory to evacuate.
*If spilled material is flammable, turn off ignition and heat sources. Don’t light Bunsen burners or turn on other switches.
*Call University of Michigan Division of Public Safety and Security (DPSS) at 911 immediately for assistance.
*Close doors to affected area.
*Post warnings to keep people from entering the area.
*Have person available that has knowledge of incident and laboratory to assist emergency personnel.

==Additional Spill Links:==
• www.oseh.umich.edu/pdf/chemspil.pdf
• http://www.oseh.umich.edu/emer-chemical.shtml.

Report all emergencies, suspicious activity, injuries, spills, and fires to the University of Michigan Division of Public Safety and Security (DPSS) by calling 911 or texting 377911. Register with the University of Michigan Emergency Alert System via Wolverine Access.

==Training of Personnel==
All personnel are required to complete the General Laboratory Safety Training session (BLS025w or equivalent) via OSEH’s My LINC website. Furthermore, all personnel shall read and fully adhere to this SOP when handling animal anesthetics.

==Certification==
I have read and understand the above SOP. I agree to contact my Supervisor or Lab manager if I plan to modify this procedure. Sign by logging in and typing <nowiki>* ~~~~</nowiki> in the list below:

*[[User:Reddj|Reddj]] ([[User talk:Reddj|talk]]) 14:44, 13 October 2016 (UTC)

* [[User:Iharvey|Iharvey]] ([[User talk:Iharvey|talk]]) 19:34, 10 November 2016 (UTC)

*[[User:Pfeiferl|Pfeiferl]] ([[User talk:Pfeiferl|talk]]) 18:09, 5 June 2017 (UTC)

* [[User:Snyderds|Snyderds]] ([[User talk:Snyderds|talk]]) 18:34, 7 June 2017 (UTC)
* [[User:Elhabbal|Elhabbal]] ([[User talk:Elhabbal|talk]]) 21:38, 12 June 2017 (UTC)
[[User:Mollyec|Mollyec]] ([[User talk:Mollyec|talk]]) 14:48, 13 June 2017 (UTC)
Prior Approval required – Is this procedure hazardous enough to warrant prior approval from the Laboratory Director? ☐ YES X NO

Laboratory Director - Dave Bridges Revision Date - 2016-10-13

Western Blotting

2017-06-12T20:03:37Z

Mollyec: /* Protocol */

==Materials==
*Transfer Buffer (200 mL Methanol, 100 mL 10X [[ Transfer Buffer ]] to final 1L volume)
*Transfer Apparatus, either Bio-Rad or Invitrogen

==Protocol==
#Run SDS-PAGE gel using [[ SDS-PAGE Running Buffer ]] and prepare diluted transfer buffer
## Use a prepared 5-12% tris gel (in the 4 degree). Remove it from the packaging, remove the white strip of tape from the bottom back, and gently pull the comb out.
##Load into gel tank. Fill with SDS page Running buffer (1X) to the fill line in the front and halfway up the back
##Load 3 microliters of protein ladder (purple), and 10 microliters of each sample into separate wells.
## Place top on tank, plug into power source and run at 125 Volts until samples and ladder reach the bottom of the gel
#Make sandwich, ensuring no bubbles between layers with black piece on bottom and layer as above. Place in apparatus so that the black sandwich touches the black transfer piece. Fill with transfer buffer.
#Transfer 4h at 75V (in cold room) or overnight at 35V (on the bench).
#Stain for total protein with Revert total protein stain (let sit for 5 minutes, after 5 minutes pour total protein stain back in bottle for later use)
#Wash twice for 5 minutes each in revert wash solution (60ml MeOH, 13.4 ml Aceditc Acid, 126.6 ml Water)
#Scan using licor for total protein, which will be used to normalize the blot
#Rinse nitrocellulose in revert reversal solution for at least 5 and no more than 10 minutes until nitrocellulose appears clear again (.2g NaOH, 60ml MeOH, 140ml Water)
#Rinse nitrocellulose in 2% BSA (2g BSA in 100ml TBST, stored in fridge) for 1 hour
#Incubate with primary antibody (check for dilution) in 1 mg/mL BSA for >1h
#Wash blot every 5 minutes for 15 min with TBST.
#Incubate with appropriate secondary antibody (10 000X) for 45min-1h (20 ml 2% BSA 1ul of both secondary antibodies, all found in fridge Ab stocks with blue dots on top)
#Wash blot every 5 minutes for 15 min with TBST.
#Rinse once or twice with double distilled water
#Prepare ECL by mixing one volume of the black bottle solution with one volume of the white bottle solution (be careful not to cross-contaminate ECL bottles with wrong solution).
#Drain excess buffer from blot and cover with ECL for about a minute
#Drain excess ECL from blot, cover with saran wrap and expose film

==If Using LiCor==
#Start -> New -> Scan Image -> Login -> Peloquin -> Password Located in Desk -> Select Dimensions -> Start Scan

[[ Category: Western Blotting ]]

High Fat Diet Treatment

2017-05-22T19:33:02Z

Mollyec: /* Diet Choices */

[[ Category: Diet ]]
[[ Category: Animal Studies ]]
[[ Category: Glucose Homeostasis ]]
[[ Category: Metabolism ]]
[[ Category: Mouse Work ]]

To place mice on a high fat diet, here is the general protocol:

For C57BL/6J mice, order so that they are 9 weeks of age on the monday of delivery. Start the diet on the Wednesday (10 days later).

==Diet Choices==

For more information about diets see [http://www.researchdiets.com/system/resources/BAhbBlsHOgZmIisyMDEyLzA0LzIwLzE0XzM5XzQwXzMxN19ESU9fU2VyaWVzLnBkZg/DIO%20Series.pdf research diets]
for caloric content in future diets, use metabolizable energy, not physiological fuel value. Metabolizable energy is more accurate as it is derived from bomb calorimetry

{| border=1
|+ Food Souces and Notes about their Composition
|-
! Food
! Vendor
! Code
! Caloric Content
! Fat
! Protein
! Sucrose
! Starch
! Notes
|-
| Normal Chow Diet
| Lab Diet
| 5L0D
| 2.91 kcal/g
| 5%
| 24%
| 3.7%
| 32%
| Provided by ULAM
|-
| High Fat Diet
| Research Diets
| D12451
| 4.73 kcal/g
| 45%
| 20%
| 17%
| 7%
| Pink
|-
| Control Diet (High Sucrose)
| Research Diets
| D12450B
| 3.85 kcal/g
| 10%
| 20%
| 35%
| 31%
| Yellow
|-
| Control Diet (High Starch)
| Research Diets
| D12450H
| 3.85 kcal/g
| 10%
| 20%
| 17%
| 44%
| Peach
|}

==Treatment Protocol==

# Start when mice are 10 weeks of age.
# For High Fat Diet mice place in cages 4 mice/cage as early as possible. Can us 5 mice/cage for normal chow/control diets.
# Re-cage (and ear tag mice if necessary) mice and replace food with ~400g food in the container. Record this weight.
# This food should last 2 weeks.
# Weigh mice at ~10 AM either weekly or bi-weekly and re-weigh food to determine food intake. Top up to 400g.
# Generally treatment lasts 12 weeks.

High Fat Diet Treatment

2017-05-22T19:31:50Z

Mollyec: /* Diet Choices */

[[ Category: Diet ]]
[[ Category: Animal Studies ]]
[[ Category: Glucose Homeostasis ]]
[[ Category: Metabolism ]]
[[ Category: Mouse Work ]]

To place mice on a high fat diet, here is the general protocol:

For C57BL/6J mice, order so that they are 9 weeks of age on the monday of delivery. Start the diet on the Wednesday (10 days later).

==Diet Choices==

For more information about diets see [http://www.researchdiets.com/system/resources/BAhbBlsHOgZmIisyMDEyLzA0LzIwLzE0XzM5XzQwXzMxN19ESU9fU2VyaWVzLnBkZg/DIO%20Series.pdf research diets]

{| border=1
|+ Food Souces and Notes about their Composition
|-
! Food
! Vendor
! Code
! Caloric Content
! Fat
! Protein
! Sucrose
! Starch
! Notes
|-
| Normal Chow Diet
| Lab Diet
| 5L0D
| 2.91 kcal/g
| 5%
| 24%
| 3.7%
| 32%
| Provided by ULAM
|-
| High Fat Diet
| Research Diets
| D12451
| 4.73 kcal/g
| 45%
| 20%
| 17%
| 7%
| Pink
|-
| Control Diet (High Sucrose)
| Research Diets
| D12450B
| 3.85 kcal/g
| 10%
| 20%
| 35%
| 31%
| Yellow
|-
| Control Diet (High Starch)
| Research Diets
| D12450H
| 3.85 kcal/g
| 10%
| 20%
| 17%
| 44%
| Peach
|}

==Treatment Protocol==

# Start when mice are 10 weeks of age.
# For High Fat Diet mice place in cages 4 mice/cage as early as possible. Can us 5 mice/cage for normal chow/control diets.
# Re-cage (and ear tag mice if necessary) mice and replace food with ~400g food in the container. Record this weight.
# This food should last 2 weeks.
# Weigh mice at ~10 AM either weekly or bi-weekly and re-weigh food to determine food intake. Top up to 400g.
# Generally treatment lasts 12 weeks.

Triglyceride Assay from Cells and Tissues

2017-05-16T15:17:51Z

Mollyec: /* Protocol */

==Materials==
* '''Homogenization Buffer''' (50 mM Tris pH 8, 5 mM EDTA, 30 mM Mannitol, PI inhibitor, can be made in bulk without the PI, PI added fresh)
* 10M KOH
* '''Chloroform/Methanol Mixture''' (2:1)
* '''Butanol Mixture''': 3 mL butanol, 1.66 mL Triton-X114, 0.33 mL Methanol
* Sigma Triglyceride Assay Kit (Cat TR0100)

==Protocol==
#Weigh out 30-50mg tissue (record weights for normalization) on dry ice into round bottom eppendorf tube (2 mL). Add one stainless steel ball bearing.
#Add 500ul Homogenization Buffer
#Homogenize with Qiagen Tissue Lyser for 3 minutes @ 25Hz for Liver/WAT or for 5 minutes @ 30Hz for muscle
#Add 12.5ul KOH
#Mix by inverting then transfewr sample to a new 1.5 mL tube. Place dirty ball bearing in ethanol (located in the fume hood)
#Add 800ul '''Chloroform/Methanol Mixture'''
#Vortex vigorously then sit at room temperature for 5 minutes
#Centrifuge for 10 minutes @ 13000G
#Transfer 400 ul of the bottom layer into a new tube
#Let evaporate overnight at room temperature
#Add'''(50ul)''' of '''Butanol Mixture'''. See Suggested Volumes for your specific tissue.
#Measure triglyceride levels using SIgma Diagnostic Kit, using 5ul of sample.
##Resuspend triglyceride and glycerol reagent with water if necessary
##Calculate how many sample you have (samples + blank + standard curve)
##Prepare reagent. You need 560ul '''(80ul)''' of glycerol reagent and 140ul '''(20ul)''' of triglyceride reagent. Make extra and combine in a Falcon tube.
##Aliquot '''100ul into a well of a 96 well plate'''
##For standards, add 0-5 and .5ul of glycerol standard
##Add 5ul of resuspended lipid to each well to start (also make a 5ul blank of the butanol mixture) and mix.
##Let sit for ~30 mins @ room temperature (or 5mins @ 37C if you are in a hurry). If using >10ul of butanol mix the solution may be cloudy. Let it settle and it should become more clear.
##Measure absorbance @ 540nm
##If any samples are A540<0.1 or above the 5ul standard the A540, then repeat with more or less lipid as required.

Western Blotting

2017-02-15T20:00:36Z

Mollyec:

==Materials==
*Transfer Buffer (200 mL Methanol, 100 mL 10X [[ Transfer Buffer ]] to final 1L volume)
*Transfer Apparatus, either Bio-Rad or Invitrogen

==Protocol==
#Run SDS-PAGE gel using [[ SDS-PAGE Running Buffer ]] and prepare diluted transfer buffer
#Make sandwich, ensuring no bubbles between layers with black piece on bottom and layer as above. Place in apparatus so that the black sandwich touches the black transfer piece. Fill with transfer buffer.
#Transfer 4h at 75V (in cold room) or overnight at 35V (on the bench).
#Stain for total protein with Revert total protein stain (let sit for 5 minutes, after 5 minutes pour total protein stain back in bottle for later use)
#Wash twice for 5 minutes each in revert wash solution (60ml MeOH, 13.4 ml Aceditc Acid, 126.6 ml Water)
#Scan using licor for total protein, which will be used to normalize the blot
#Rinse nitrocellulose in revert reversal solution for at least 5 and no more than 10 minutes until nitrocellulose appears clear again (.2g NaOH, 60ml MeOH, 140ml Water)
#Rinse nitrocellulose in 2% BSA (2g BSA in 100ml TBST, stored in fridge) for 1 hour
#Incubate with primary antibody (check for dilution) in 1 mg/mL BSA for >1h
#Wash blot every 5 minutes for 15 min with TBST.
#Incubate with appropriate secondary antibody (10 000X) for 45min-1h (20 ml 2% BSA 1ul of both secondary antibodies, all found in fridge Ab stocks with blue dots on top)
#Wash blot every 5 minutes for 15 min with TBST.
#Rinse once or twice with double distilled water
#Prepare ECL by mixing one volume of the black bottle solution with one volume of the white bottle solution (be careful not to cross-contaminate ECL bottles with wrong solution).
#Drain excess buffer from blot and cover with ECL for about a minute
#Drain excess ECL from blot, cover with saran wrap and expose film

==If Using LiCor==
#Start -> New -> Scan Image -> Login -> Peloquin -> Password Located in Desk -> Select Dimensions -> Start Scan

[[ Category: Western Blotting ]]

Western Blotting

2017-02-03T20:40:54Z

Mollyec:

==Materials==
*Transfer Buffer (200 mL Methanol, 100 mL 10X [[ Transfer Buffer ]] to final 1L volume)
*Transfer Apparatus, either Bio-Rad or Invitrogen

==Protocol==
#Run SDS-PAGE gel using [[ SDS-PAGE Running Buffer ]] and prepare diluted transfer buffer
#Make sandwich, ensuring no bubbles between layers with black piece on bottom and layer as above. Place in apparatus so that the black sandwich touches the black transfer piece. Fill with transfer buffer.
#Transfer 4h at 75V (in cold room) or overnight at 35V (on the bench).
#Stain for total protein with Revert total protein stain (let sit for 5 minutes, after 5 minutes pour total protein stain back in bottle for later use)
#Wash twice for 5 minutes each in revert wash solution (60ml MeOH, 13.4 ml Aceditc Acid, 126.6 ml Water)
#Scan using licor for total protein, which will be used to normalize the blot
#Rinse nitrocellulose in revert reversal solution for at least 5 and no more than 10 minutes until nitrocellulose appears clear again (.2g NaOH, 60ml MeOH, 140ml Water)
#Rinse nitrocellulose in 2% BSA (in TBST) for 1 hour
#Incubate with primary antibody (check for dilution) in 1 mg/mL BSA for >1h (20 ml 2% BSA 1ul of both secondary antibodies)
#Wash blot every 5 minutes for 15 min with TBST.
#Incubate with appropriate secondary antibody (10 000X) for 45min-1h (20 ml 2% BSA 1ul of both secondary antibodies)
#Wash blot every 5 minutes for 15 min with TBST.
#Rinse once or twice with double distilled water
#Prepare ECL by mixing one volume of the black bottle solution with one volume of the white bottle solution (be careful not to cross-contaminate ECL bottles with wrong solution).
#Drain excess buffer from blot and cover with ECL for about a minute
#Drain excess ECL from blot, cover with saran wrap and expose film

==If Using LiCor==
#Start -> New -> Scan Image -> Login -> Peloquin -> Password Located in Desk -> Select Dimensions -> Start Scan

[[ Category: Western Blotting ]]

Western Blotting

2017-02-03T14:47:39Z

Mollyec:

==Materials==
*Transfer Buffer (200 mL Methanol, 100 mL 10X [[ Transfer Buffer ]] to final 1L volume)
*Transfer Apparatus, either Bio-Rad or Invitrogen

==Protocol==
#Run SDS-PAGE gel using [[ SDS-PAGE Running Buffer ]] and prepare diluted transfer buffer
#Make sandwich, ensuring no bubbles between layers with black piece on bottom and layer as above. Place in apparatus so that the black sandwich touches the black transfer piece. Fill with transfer buffer.
#Transfer 4h at 75V (in cold room) or overnight at 35V (on the bench).
#Stain for total protein with Revert total protein stain (let sit for 5 minutes, after 5 minutes pour total protein stain back in bottle for later use)
#Wash twice for 5 minutes each in revert wash solution (60ml MeOH, 13.4 ml Aceditc Acid, 126.6 ml Water)
#Scan using licor for total protein, which will be used to normalize the blot
#Rinse nitrocellulose in revert reversal solution for at least 5 and no more than 10 minutes until nitrocellulose appears clear again (.2g NaOH, 60ml MeOH, 140ml Water)
#Rinse nitrocellulose in 2% BSA (in TBST) for 1 hour
#Incubate with primary antibody (check for dilution) in 1 mg/mL BSA for >1h
#Wash blot every 5 minutes for 15 min with TBST.
#Incubate with appropriate secondary antibody (10 000X) for 45min-1h
#Wash blot every 5 minutes for 15 min with TBST.
#Rinse once or twice with double distilled water
#Prepare ECL by mixing one volume of the black bottle solution with one volume of the white bottle solution (be careful not to cross-contaminate ECL bottles with wrong solution).
#Drain excess buffer from blot and cover with ECL for about a minute
#Drain excess ECL from blot, cover with saran wrap and expose film

==If Using LiCor==
#Start -> New -> Scan Image -> Login -> Peloquin -> Password Located in Desk -> Select Dimensions -> Start Scan

[[ Category: Western Blotting ]]

Western Blotting

2017-02-03T14:46:45Z

Mollyec:

==Materials==
*Transfer Buffer (200 mL Methanol, 100 mL 10X [[ Transfer Buffer ]] to final 1L volume)
*Transfer Apparatus, either Bio-Rad or Invitrogen

==Protocol==
#Run SDS-PAGE gel using [[ SDS-PAGE Running Buffer ]] and prepare diluted transfer buffer
#Make sandwich, ensuring no bubbles between layers with black piece on bottom and layer as above. Place in apparatus so that the black sandwich touches the black transfer piece. Fill with transfer buffer.
#Transfer 4h at 75V (in cold room) or overnight at 35V (on the bench).
#Stain for total protein with Revert total protein stain (let sit for 5 minutes, after 5 minutes pour total protein stain back in bottle for later use)
#Wash twice for 5 minutes each in revert wash solution (60ml MeOH, 13.4 ml Aceditc Acid, 126.6 ml Water)
#Scan using licor for total protein, which will be used to normalize the blot
#Rinse nitrocellulose in revert reversal solution for at least 5 and no more than 10 minutes until nitrocellulose appears clear again (.2g NaOH, 60ml MeOH, 140ml Water)
#Rinse nitrocellulose in 2% BSA for 1 hour
#Carefully remove blot, stain with Ponceau solution and rinse with TTBS until all the red is washed off
#Block with 2% BSA in [[ TBST ]] or 5% skim milk powder in TBST for >1h
#Incubate with primary antibody (check for dilution) in 1 mg/mL BSA for >1h
#Wash blot every 5 minutes for 15 min with TBST.
#Incubate with appropriate secondary antibody (10 000X) for 45min-1h
#Wash blot every 5 minutes for 15 min with TBST.
#Rinse once or twice with double distilled water
#Prepare ECL by mixing one volume of the black bottle solution with one volume of the white bottle solution (be careful not to cross-contaminate ECL bottles with wrong solution).
#Drain excess buffer from blot and cover with ECL for about a minute
#Drain excess ECL from blot, cover with saran wrap and expose film

==If Using LiCor==
#Start -> New -> Scan Image -> Login -> Peloquin -> Password Located in Desk -> Select Dimensions -> Start Scan

[[ Category: Western Blotting ]]

SOP - Vacuum Pumps

2016-10-31T16:18:47Z

Mollyec:

[[Category: SOP]]
[[Category: Lab Safety]]

==Description==
This standard operating procedure outlines the handling and use of vacuum pumps. In accordance with this document, laboratories should use appropriate controls, personal protective equipment, and disposal techniques when working with vacuum pumps.

Vacuum pumps are commonly used in a variety of experimental setups to remove air and other vapors from a vessel or system. Applications that use vacuum pumps include rotary evaporators, vacuum ovens, drying manifolds, freeze-dryers, aspirators, desiccators, and filtration equipment. Many vacuum pumps use oil which can be easily contaminated and result in pump damage and hazards.

==Potential Hazards==
Vacuum pumps can pose chemical, mechanical, electrical, and fire hazards. Chemical exposure can occur from improper installing, trapping, and exhausting or from off gassing of contaminated pump oil. Mechanical hazards can occur from the moving parts of the vacuum pump. Fire can result if the pump malfunctions or overheats and ignites nearby combustible materials. Electrocution can occur from faulty or defective switches and wiring.

==Engineering Controls==
Vacuum pumps used to evacuate systems containing toxic, volatile, or corrosive substances must be vented to the building exhaust ventilation system. Whenever possible locate pump in vacuum pump cabinet or a ventilated cabinet. Ensure that pumps have belt guards. Traps such as a cold trap should be used in line with high vapor loads to minimize the amount of volatile chemicals being evacuated and reaching the pump oil. Ensure the cold trap is appropriate for situation and follows all manufacturer and safety guidelines.

==Work Practice Controls==
===General:===
* Place pump on spill tray to contain oil spills if the equipment fails.
* Shield any glassware under vacuum.
===Physical:===
* Ensure all vacuum pump belt systems are guarded.
* Electrical cords and switches must be free from defects.
* Avoid placing pumps in an unventilated and enclosed receptacle.
* Do not operate pumps near combustible materials.
* Use correct vacuum tubing and replace old tubing.
===Chemical:===
* Use engineering controls to avoid exposure.
* Ensure pump oil is compatible with vapors that will pass through the pump (i.e. avoid hydrocarbon pump oil and oxidizing gases/vapors).
* Close valve between vessel and pump before turning off pump to avoid introducing oil into system.
* Ensure gases or vapors will not damage the pump.
* If cold traps are used, check for blockage.
* Check oil levels and change oil when necessary. Change oil when it begins to turn a dark brown color.
* An oil mist separator can be used to prevent oil loss.

==Personal Protective Equipment (PPE)==
Wear safety glasses, lab coat, long pants, close-toed shoes and gloves when performing all vacuum operations.

==Transportation and Storage==
Vacuum pumps must be stored on spill trays to prevent oil spills. Vacuum pump areas may be insulated for noise, if necessary.

==Waste Disposal==
All used vacuum pump oils must be dispose of through OSEH.

Do not dispose of chemical wastes by dumping them down a sink, flushing in a toilet or discarding in regular trash containers, unless authorized by OSEH Hazardous Materials Management (HMM). Contact OSEH-HMM at (734) 763-4568 for waste containers, labels, manifests, waste collection and for any questions regarding proper waste disposal. Also refer to OSEH’s Hazardous Waste webpage for more information.

==Exposures/Unintended Contact==

If the employee is in need of emergency medical attention, call 911 immediately.

Contact OSEH for advice on symptoms of chemical exposure, or assistance in performing an exposure assessment.

Report all work related accidents, injuries, illnesses or exposures to WorkConnections within 24 hours by completing and submitting the Illness and Injury Report Form. Follow the directions on the WorkConnections website Forms Instructions to obtain proper medical treatment and follow-up.

Complete the OSEH Laboratory Incident and Near-Miss Report form.

===TREATMENT FACILITIES:===
===U-M Occupational Health Services -- Campus Employees===
Mon-Fri 7:30 am - 4:30 pm
After hours - go to UM Hospital Emergency Dept. – Urgent Care Clinic
C380 Med Inn building
1500 East Medical Center Drive, Ann Arbor (734) 764-8021
===University Health Services -- University students (non-life threatening conditions)===
Mon-Fri 8 am – 4:30 pm, Sat 9 am – 12 pm
Contact for current hours as they may vary
207 Fletcher Street, Ann Arbor (734) 764-8320
===UMHS Emergency Department -- after clinic hours or on weekends===
1500 East Medical Center Drive, Ann Arbor, (734) 936-6666
Click here for additional accident and injury reporting information.

==Spill Procedure==
A minor (small) chemical spill is one that the laboratory staff is capable of handling safely without the assistance of safety and emergency personnel, i.e., less than 1 Gallon or 3.5 Liters. A major/large chemical spill requires active assistance from emergency personnel.

For small oil spills, treat the oil as a hazardous chemical spill. Use appropriate absorbent and dispose of as hazardous waste. For large oil spills contact OSEH-HMM at (734) 763-4568.

* When a spill occurs, personal safety should always come first.
* Alert and clear everyone in the immediate area where the spill occurred.

Additional Spill Response Steps:

===MINOR CHEMICAL SPILL===
* Alert people in immediate area of spill.
* If spilled material is flammable, turn off ignition and heat sources. Don’t light Bunsen burners or turn on other switches.
* Open outside windows, if possible.
* Wear protective equipment, including safety goggles, gloves and long-sleeve lab coat.
* Avoid breathing vapors from spill.
* Confine spill to as small an area as possible.
* Do not wash spill down the drain.
* Use appropriate spill kits/sorbents to absorb spill. Collect contaminated materials and residues and place in container. Contact OSEH-HMM (734) 763-4568 for proper disposal.
* Clean spill area with water.

===MAJOR CHEMICAL SPILL===
* Attend to injured or contaminated persons and remove them from exposure.
* Alert people in the laboratory to evacuate.
* If spilled material is flammable, turn off ignition and heat sources. Don’t light Bunsen burners or turn on other switches.
* Call University of Michigan Division of Public Safety and Security (DPSS) at 911 immediately for assistance.
* Close doors to affected area.
* Post warnings to keep people from entering the area.
* Have person available that has knowledge of incident and laboratory to assist emergency personnel.

Additional Spill Links:
* www.oseh.umich.edu/pdf/chemspil.pdf
* http://www.oseh.umich.edu/emer-chemical.shtml.

Report all emergencies, suspicious activity, injuries, spills, and fires to the University of Michigan Division of Public Safety and Security (DPSS) by calling 911 or texting 377911. Register with the University of Michigan Emergency Alert System via Wolverine Access.

==Training of Personnel==
All personnel are required to complete the General Laboratory Safety Training session (BLS025w or equivalent) via OSEH’s My LINC website. Furthermore, all personnel shall read and fully adhere to this SOP when working with vacuum pumps.

Certification
I have read and understand the above SOP. I agree to contact my Supervisor or Lab Manager if I plan to modify this procedure. Sign by logging in and typing <nowiki>* ~~~~</nowiki> in the list below:

Prior Approval required – Is this procedure hazardous enough to warrant prior approval from the Laboratory Director? ☐ YES X NO

*[[User:Mollyec|Mollyec]] ([[User talk:Mollyec|talk]]) 16:18, 31 October 2016 (UTC)

Laboratory Director - Dave Bridges Revision Date - 2016-10-19