2017-10-19T19:41:01Z

Iharvey:

__NOTOC__
[[ Category: Cloning ]]
[[ Category: Molecular Biology ]]
[[ Category: Genome Editing ]]
[[ Category: CRISPR ]]

For design considerations see [[ Designing and Generating CRISPR-Cas Mutants ]]

=Cloning=
* First digest vector by adding to a PCR tube (or use a frozen, pre-digested vector):
** 2 ug of pX335/pX459
** 1uL of 10X NEB Buffer 2.1
** 1 uL of CIAP
** 1 uL of BbsI
** water up to 10 uL
** Digest for 1h in, gel purify the fragment from an agarose gel, using the QIAEX II Kit and check the concentration by nanodrop.
* Next prepare the insert by annealing and phosphorylating the primers in a PCR tube:
** 4.5 uL of water
** 1 uL of a 100 uM stock of each oligo
** 2 uL of 5X ligase buffer
** 1 uL of T4 PNK
** Incubate at 37C for 30 mins then 95C for 5 mins to heat inactivate PNK then ramp down to 25C at 5C/min to allow the oligos to anneal, leave at room temperature
** Dilute the annealed oligos 250X in water (2 uL + 498 uL of water)
* Combine the ligation mixture in an eppendorf tube:
** 50 ng of vector
** 3 uL of annealed insert or '''water as a blank'''
** 2 uL of 5X ligation buffer
** 1 uL of T4 DNA Ligase
** Water to 10 uL
* Incubate for 10 min at RT
* Transform 2uL of this into competent cells (see [[Transformation of Bacteria]]), plating the entire transformation.

===Verification===
* Miniprep the transformed clones and digest 1 ug of purified plasmid for 1h with AgeI and BbsI in NEB Buffer 1.1 and then run on an agarose gel. Empty vectors will yield a ~1kb fragment, wheras vectors with insert will only be cut once. This is because the cloning removes the BbsI site.
* Send clones with insert for sequencing with the hU6 sequencing primer. Go to http://bridgeslab.sph.umich.edu/protocols/index.php/Submitting_Plasmids_for_Sequencing for further details. Align those clones with the empty vector full sequence or the hU6 sequencing of the empty vector, available at http://www.addgene.org/42335/sequences/. Doublecheck that your insert is correct, and is oriented in the correct manner.

Submitting Plasmids for Sequencing

2017-10-19T19:38:50Z

Iharvey:

Submitting Plasmids for Sequencing

2017-10-19T19:35:10Z

Iharvey:

*Go to https://client-seqcore.brcf.med.umich.edu/login.cgi?exp=1
**PI login: Bridges
**Dave must add you to the lab under his account, he will give you your username and the password needed
*Typically we will select Sanger Sequencing (the only low throughput option)
*Select 'Submit samples/plates'
*Select the appropriate short-code from the drop down menu at the top of this page
*Generally choose 'One-at-a-time Sample Submission' unless you have a lot
*From here enter the following for each section:
**The sample name (the sample name that mean something to you regarding the sequence)
**No discounts
**Mini-Prep Plasmid
**Whatever vector you used for this plasmid
**Size of the insert you are sequencing
**Custom Primers
**Do not save
**Do not rush
*Save and repeat with your other samples
*Once you have finished adding your samples ID codes for each sample will be generated and these are the IDs you need to use for the tubes you are submitting, not the sample names you made
**Make sure to write these IDs very clearly
**A submission ID will also be generated and you need to know this when you drop your samples off so they can find your sequence request in their system
*Plasmids need to be diluted to a concentration of ~50ng/ul and total volume should be 10ul and should be labeled with the generated sample ID
*In addition to your samples you will need to provide an aliquot of the primer
**Volume is 10ul/sample you submit
**Should be labeled as the range of sample IDs generated and P1 (example: xxx-xxxP1), if you have more than one primer write xxx-xxxP2 and so on
*These should be submitted in 1.5mL tubes
*Go to https://seqcore.brcf.med.umich.edu/sample-dropoff-locations for locations and drop off times

Submitting Plasmids for Sequencing

2017-10-19T19:26:19Z

Iharvey: Created page with "*Plasmids need to be diluted to a concentration of ~50ng/ul and total volume should be 10ul *These should be submitted in 1.5mL tubes *Go to https://client-seqcore.brcf.med.um..."

*Plasmids need to be diluted to a concentration of ~50ng/ul and total volume should be 10ul
*These should be submitted in 1.5mL tubes
*Go to https://client-seqcore.brcf.med.umich.edu/login.cgi?exp=1
**PI login: Bridges
**Dave must add you to the lab under his account, he will give you your username and the password needed
*Typically we will select Sanger Sequencing (the only low throughput option)
*Select 'Submit samples/plates'
*Select the appropriate short-code from the drop down menu at the top of this page
*Generally choose 'One-at-a-time Sample Submission' unless you have a lot
*From here enter the following for each section:
**The sample name (the sample name that mean something to you regarding the sequence)
**No discounts
**Mini-Prep Plasmid
**Whatever vector you used for this plasmid
**Size of the insert you are sequencing
**Custom Primers
**Do not save
**Do not rush
*Save and repeat with your other samples
*Go to ... for locations and drop off times

Western Blotting

2017-08-11T15:52:47Z

Iharvey: /* 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 (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!
#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 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 ]]

Bradford Assay

2017-08-08T15:52:46Z

Iharvey: /* Protocol */

==Materials==
*BioRad Protein Assay Dye Reagent Concentrate cat#500-0006
*Disposable Plastic Cuvette
*0.1mg/mL BSA in H20 as standard

==Protocol==
Cuvette Bradford Assay
#Dilute reagent 5X in water, stable for 2-3 weeks
#Pipet 1 mL into disposable plastic cuvette
#Add 1-10 uL of protein sample, cover with parafilm and mix
#Let sit 5-10 min to react
#Set spectrophotometer as follows:
##Go to protein assay then Bradford assay
##Set formula, then select more
##Set b=0.045 (or determine slope)
##Set dilution to be 1/vol (ie 0.1 for 10 uL)
##Blank then measure samples, absorbance must be less than 0.9
##Print (hit Recall, then enter, then print) and attach to experiment

Protein Lysate Bradford Assay
#Dilute reagent 5X in water, stable for 2-3 weeks
#In a 96 well plate, dilute sample 20X (190ul H2O, 10ul Sample)--this dilution factor is tissue-dependent, only need to dilute fat ~5x
#Add 5ul of 20x diluted sample to 100ul of reagent in well
#Run "Bradford Assay Protocol" on Plate Reader

==Reference==
*Wikipedia: [[wikipedia:Bradford_protein_assay|Bradford Protein Assay]]
*PMID 942051

Bradford Assay

2017-08-08T15:51:15Z

Iharvey: /* Protocol */

==Materials==
*BioRad Protein Assay Dye Reagent Concentrate cat#500-0006
*Disposable Plastic Cuvette
*0.1mg/mL BSA in H20 as standard

==Protocol==
Cuvette Bradford Assay
#Dilute reagent 5X in water, stable for 2-3 weeks
#Pipet 1 mL into disposable plastic cuvette
#Add 1-10 uL of protein sample, cover with parafilm and mix
#Let sit 5-10 min to react
#Set spectrophotometer as follows:
##Go to protein assay then Bradford assay
##Set formula, then select more
##Set b=0.045 (or determine slope)
##Set dilution to be 1/vol (ie 0.1 for 10 uL)
##Blank then measure samples, absorbance must be less than 0.9
##Print (hit Recall, then enter, then print) and attach to experiment

Protein Lysate Bradford Assay
#Dilute reagent 5X in water, stable for 2-3 weeks
#In a 96 well plate, dilute sample 20X (190ul H2O, 10ul Sample)
#Add 5ul of sample to 100ul of reagent in well
#Run "Bradford Assay Protocol" on Plate Reader

==Reference==
*Wikipedia: [[wikipedia:Bradford_protein_assay|Bradford Protein Assay]]
*PMID 942051