Bridges Lab Protocols - User contributions [en]

Fugene Transfection of 293T/COS Cells

2009-06-10T14:07:43Z

Jpecherer: /* Protocol */

==Protocol==
#Warm OptiMEM, COS-FBS Media and PBS -/-.
#Calculate amount of Fugene needed.
##Per ug of DNA need 3 uL Fugene.
##Per uL of Fugene need 16 uL of OptiMEM.
#Add Fugene to OptiMEM, incubate ~5 min.
#Add required amount of DNA to eppendorf tubes.
#Add 51 uL OptiMEM/Fugene per ug DNA to each DNA tube.
#Incubat 5-20 min.
#Split confluent cells 2-3X into fresh dishes as follows:
##Wash confluent COS cell plate(s) twice with 10 mL D-PBS -/-
##Add 1 mL Trypsin solution (0.05%) and incubate at 37C until cells are detached
##Add 25 mL COS/FBS
##Aliquot cells into wells as required (1 mL per 12well, 2 mL per 6 well)
#Add DNA/Fugene/DMEM to cells
#Leave mixture on Cells for 24-48h to allow protein to accumulate

Surface Plasmon Resonance - Protein Lipid Interactions

2009-06-10T14:07:02Z

Jpecherer: /* Materials */

==Materials==
*L1 Sensor Chip (Biacore)
*10x Dioleylphosphatidylcholine (Sigma) dissolved in 1:1 chloroform:methanol with 0.1% HCl
*10x PIPx of interest (Avanti or Echelon) dissolved in 1:1 chloroform:methanol with 0.1% HCl
*HBS-N (25mM HEPES, pH 7.4, 150 mM NaCl)(6.25mL Hepes (1M), 9.38mL NaCl (4M)) Total volume 250mL.
*1M NaOH
*pH strips
*Water bath sonicator set to 40C
*Avanti MiniExtruder
*1% beta octylglucoside
*0.5% SDS
*30% ethanol
*100mM NaOH

==Preparation of Liposomes==
#Combine DOPC + C16 lipid (or DOPC alone) at a ratio of 97:3 and dry under nitrogen (15uL PC or 14.5uL PC + 0.5 uL PI)
#Resuspend to 10 mM total lipid with HBS-N (100 uL). Vortex thoroughly and sonicate in water bath
#Correct pH to 7.4 using pH paper and 0.6uL aliquots of 1M NaOH
#Freeze thaw in a water bath sonicator at 40C and liquid nitrogen 8 times.
#Pass 10X through a polycarbonate filter using an Avanti MiniExtruder at Room Temperature.
*Rinse syringe with milliQ water.
*Wet filter paper and put the filter on the block.
*Place PC filter on block.
*Tightly attach syringe and place on blcok.
*Pass 250uL of milliQ water through the filger ~5 times.
*Discard the water and inject the sample through the filter 10 times.

==Preparation of Surface==
#Wash all four surfaces at 10 uL/min and 25C with HBS-N
#Inject 20 uL of 1% beta-octylglucoside
#Inject 20 uL of 0.5% SDS
#Inject 10 uL of 1% beta-octylglucoside
#Inject 10 uL of 30% ethanol
#Inject 70 uL of extruded lipid suspension at 10uL/min (should see an increase of 5000-10000RU) to desired well (start with lane 4 and move to lane 1-PC only, lipids can migrate so load in a way to minimize contamination affecting results)
#Wash with 20 uL of 0.1M NaOH

==Analysis of Sample==
#Inject protein samples adjusting contact time as necessary to reach saturation (typically 400s per injection)
#Inject 20 uL 0.1M NaOH between samples
#For Kd determination, inject buffer 2-3x first to get a blank reading

==Reference==
<pubmed>16829131</pubmed>

Surface Plasmon Resonance - Protein Lipid Interactions

2009-06-10T14:06:12Z

Jpecherer: /* Preparation of Liposomes */

==Materials==
*L1 Sensor Chip (Biacore)
*Dioleylphosphatidylcholine (Sigma) dissolved in 1:1 chloroform:methanol with 0.1% HCl
*PIPx of interest (Avanti or Echelon) dissolved in 1:1 chloroform:methanol with 0.1% HCl
*HBS-N (25mM HEPES, pH 7.4, 150 mM NaCl)(6.25mL Hepes (1M), 9.38mL NaCl (4M)) Total volume 250mL.
*1M NaOH
*pH strips
*Water bath sonicator set to 40C
*Avanti MiniExtruder
*1% beta octylglucoside
*0.5% SDS
*30% ethanol
*100mM NaOH

==Preparation of Liposomes==
#Combine DOPC + C16 lipid (or DOPC alone) at a ratio of 97:3 and dry under nitrogen (15uL PC or 14.5uL PC + 0.5 uL PI)
#Resuspend to 10 mM total lipid with HBS-N (100 uL). Vortex thoroughly and sonicate in water bath
#Correct pH to 7.4 using pH paper and 0.6uL aliquots of 1M NaOH
#Freeze thaw in a water bath sonicator at 40C and liquid nitrogen 8 times.
#Pass 10X through a polycarbonate filter using an Avanti MiniExtruder at Room Temperature.
*Rinse syringe with milliQ water.
*Wet filter paper and put the filter on the block.
*Place PC filter on block.
*Tightly attach syringe and place on blcok.
*Pass 250uL of milliQ water through the filger ~5 times.
*Discard the water and inject the sample through the filter 10 times.

==Preparation of Surface==
#Wash all four surfaces at 10 uL/min and 25C with HBS-N
#Inject 20 uL of 1% beta-octylglucoside
#Inject 20 uL of 0.5% SDS
#Inject 10 uL of 1% beta-octylglucoside
#Inject 10 uL of 30% ethanol
#Inject 70 uL of extruded lipid suspension at 10uL/min (should see an increase of 5000-10000RU) to desired well (start with lane 4 and move to lane 1-PC only, lipids can migrate so load in a way to minimize contamination affecting results)
#Wash with 20 uL of 0.1M NaOH

==Analysis of Sample==
#Inject protein samples adjusting contact time as necessary to reach saturation (typically 400s per injection)
#Inject 20 uL 0.1M NaOH between samples
#For Kd determination, inject buffer 2-3x first to get a blank reading

==Reference==
<pubmed>16829131</pubmed>

Colocalization

2009-06-03T20:44:01Z

Jpecherer: Created page with '#Open ImageJ Software #Prior to performing colocalisation analysis save cell images as experiment files #Open an experiment file in ImageJ by File->Open->Select File #Select Fi...'

#Open ImageJ Software
#Prior to performing colocalisation analysis save cell images as experiment files
#Open an experiment file in ImageJ by File->Open->Select File
#Select File and create composite by Image->color>create composite
#Select File and split by Image->color->Split
#Select each image and change to 8-bit greyscale by Image->type->8bit
#Assess colocalisation by Plugins->Colocalisation analysis

'''Assessment'''

----

'''Rr'''

This is the Pearson’s correlation coefficient. Zero-zero pixels are not included in this calculation.

This is a popular method of quantifying correlation in many fields of research from psychology to economics. In many forms of correlation analysis the values for Pearson’s will range from 1 to -1. A value of 1 represents perfect correlation; -1 represents perfect exclusion and zero represents random localisation. However, this is not the case for images. While perfect correlation gives a value of 1, perfect exclusion does not give a value of -1. Low (close to zero) and negative values for Pearson’s correlation coefficient for fluorescent images can be difficult to interpret. However, a value close to 1 does indicate reliable colocalisation.

----

'''R'''

This is Mander’s Overlap coefficient. This is easier than the Pearson’s coefficient to comprehend. It ranges between 1 and zero with 1 being high-colocalisation, zero being low. However, the number of objects in both channel of the image has to be more or less equal.

----

'''Slope'''
Slope of the line represents the "red to green" ratio, as a measure of both image intensity and colocalization. Ideally, the slope should be equal to 1 (y=x), however, it is more likely that one of the immunostained colors will be darker than the other causing the slope to tend more towards that axis. Good colocalization will give a scatterplot which is best fit by a linear curve, where the slope of this curve is representative of the ratio of immunostained colors.

See following link for more information:
[http://www.uhnresearch.ca/facilities/wcif/imagej/colour_analysis.htm]

Main Page

2009-06-03T20:29:18Z

Jpecherer: /* Cell and Tissue Culture */

==Cell and Tissue Culture==
*[[Splitting Cells]]
*[[Differentiation of 3T3-L1 Cells]]
*[[Electroporation of 3T3-L1 Adipocytes]]
*[[Immunofluoresence]]
*[[3T3-L1 Adipocyte Fractionation]]
*[[Preparing Cell Lysates]]
*[[Glucose Uptake Assay]]
*[[Luciferase Assay]]
*[[Colocalization]]

==Cloning and Molecular Biology==
*[[Transformation of Bacteria]]
*[[Mutagenesis]]
*[[PCR Amplification of DNA]]
*[[Restriction Enzyme Based Cloning]]
**[[Restriction Enzyme Based Cloning - Ordering Primers]]
*[[TOPO Cloning]]
*[[Preparing an Agarose Gel]]
*[[Cesium Chloride Preparation of DNA]]

==Protein Analysis==
*[[Western Blotting]]
*[[Surface Plasmon Resonance - Protein Lipid Interactions]]
===Protein Quantification===
*[[Bradford Assay]]
*[[Quantification by Absorbance at 280nm]]
*[[Affinity Purificatoin of Antibodies (Coupling to Activated CH Sepharose 4B)]]

===Protein Purification===
*[[French Press]]
*[[Purification of GST Fusion Proteins]]
*[[Determining Percent Purity]]

==Transcriptional Analysis==
*[[Real Time PCR From Cell Culture]]
*[[Harvesting RNA from Cells grown in monolayer]]

==Mouse Protocols==
===Genotyping===
*[[Ear Tagging and Tail Clipping]]
*[[DNA Preparation from Tail Clip]]
*[[PCR Analysis of Tail DNA]]
===Metabolic Measurements===
*[[Glucose Tolerance Test]]

Affinity Purificatoin of Antibodies (Coupling to Activated CH Sepharose 4B)

2009-05-20T13:52:26Z

Jpecherer:

'''Materials'''
#0.1M NaBicarbonate (0.8401g in 100mL ddH2O) and 0.5MNaCl (2.922g in 100mL ddH2O)at pH 8.0.
#50mM Sodium Acetate (0.41g in 100mL ddH2O)at pH 4.0
#50mM Tris (0.61g in 100mL ddH2O) and .5M NaCl (2.922g in 100mL ddH2O) at pH 8.0
#0.4M glycine (3.004g in 100mL ddH2O)at pH 1.8.

'''Procedure'''
Protein must be extensively dialysed in PPBS to remove any amine group from the sample.
#Swell 0.5g of CH Sepharose 4B in 200 mL of ice cold 1 mM HCl for 15 minutes. Gives ~1.5 mL of Sepharose.
#Wash separose with coupling buffer (1.0M NaHCO3), 0.5 M NaCl, pH 8.0)
#Dissolve protein ligand (~1mg) in 6 mL of coupling buffer.
#Mix sepharose and ligand end over end at room temperature for 2-4 hours.
#Retain flow through for analysis and wash sepharose with coupling buffer.
#Block Sepharose with 1 M Tris/HCl, pH 8.0 for 1 hour end over end at room temperature. Alternatively block overnight at 4oC.
#Wash sepharose with 3 cycles of 50mM sodium acetate, 0.5 M NaCl, pH 4.0 the 50 mM Tris/HCl, 0.5 M NaCl, pH 8.0.
#Store in coupling buffer with 0.02% sodium azide.

'''Coupling Efficiency'''
Add 10 uL of flow through to 1 mL of Bradford's Reagent.
Coupling % should be between 70 and 80%.
{| border="1" cellspacing="0" cellpadding="5" align="center"
| Average OD595
|
|-
| mg/mL
|
|-
|Total Volume
|
|-
|Total mg
|
|-
|% Coupling
|
|-
|}

For Peptides use 5 mg in 1 ml of coupling buffer. Check pH has not changed. Read OD241 before and after coupling for coupling efficiency.

'''Affinity Purification'''
#Dialysis of 10 mL sera against PBS for 1-2 hours at 4oC.
#Wash Sepharose with 2 column volumes of 50 mM Tris/HCl, pH 7.5 with 0.5 M nACl then 50 mM Tris/HCl, pH7.5.
#Dilute sera with equal volume of 50 mM Tris/HCl, pH 7.5.
#Load onto column end over end for 2-4 hours at 4oC.
#If column flow through needs to be kept store at -80oC.
#Wash sepharose with 50 mM Tris/HCl, pH 7.5 with 0.5 M NBaCl.
#Pack into Bio-rad plastic column. Wash until eluent is clean.
#Elute antibodies with 100 mM glycine pH 1.8. Collect 10 x 1 mL fractoins. Bring pH of antibodies back to neutral by adding ~250 uL of 1 M Tris/HCl, pH 8.0.
#Assay for protein and pool the protein peak.
#Dialysis against 1 L of PBS for 2-4 hours and 2 L of PBS overnight at 4oC.
#Concentrate sample, assay for protein and aliquot antibodies. Store at -80oC.
#Store column in 50 mM Tris/HCl, pH 7.5 with sodium azide at 4oC.

Affinity Purificatoin of Antibodies (Coupling to Activated CH Sepharose 4B)

2009-05-20T13:17:11Z

Jpecherer:

Affinity Purificatoin of Antibodies (Coupling to Activated CH Sepharose 4B)

2009-05-19T20:30:59Z

Jpecherer: Created page with ''''Materials''' #0.1M NaBicarbonate (0.8401g in 100mL ddH2) and 0.5MNaCl (2.922g in 100mL ddH2)pH 8.0.'

'''Materials'''
#0.1M NaBicarbonate (0.8401g in 100mL ddH2) and 0.5MNaCl (2.922g in 100mL ddH2)pH 8.0.

Main Page

2009-05-19T20:27:21Z

Jpecherer:

==Cell and Tissue Culture==
*[[Splitting Cells]]
*[[Differentiation of 3T3-L1 Cells]]
*[[Electroporation of 3T3-L1 Adipocytes]]
*[[Immunofluoresence]]
*[[3T3-L1 Adipocyte Fractionation]]
*[[Preparing Cell Lysates]]
*[[Harvesting RNA from Cells grown in monolayer]]

==Cloning and Molecular Biology==
*[[Transformation of Bacteria]]
*[[Mutagenesis]]
*[[PCR Amplification of DNA]]
*[[Restriction Enzyme Based Cloning]]
**[[Restriction Enzyme Based Cloning - Ordering Primers]]
*[[TOPO Cloning]]
*[[Preparing an Agarose Gel]]

==Protein Analysis==
*[[Western Blotting]]
*[[Surface Plasmon Resonance - Protein Lipid Interactions]]
===Protein Quantification===
*[[Bradford Assay]]
*[[Quantification by Absorbance at 280nm]]
*[[Affinity Purificatoin of Antibodies (Coupling to Activated CH Sepharose 4B)]]

===Protein Purification===
*[[French Press]]
*[[Purification of GST Fusion Proteins]]
*[[Determining Percent Purity]]

==Transcriptional Analysis==
*[[Real Time PCR From Cell Culture]]
*[[Harvesting RNA from Cells grown in monolayer]]

==Mouse Protocols==
===Genotyping===
*[[Ear Tagging and Tail Clipping]]
*[[DNA Preparation from Tail Clip]]
*[[PCR Analysis of Tail DNA]]
===Metabolic Measurements===
*[[Glucose Tolerance Test]]

Harvesting RNA from Cells grown in monolayer

2009-05-12T15:34:29Z

Jpecherer:

'''Loading the Column'''
#Harvest cells grown in a monolayer (do not use more than 107 cells): Cells can be either lysed directly in the cell-culture vessel (up to 10 cm diameter) or trypzinized and collected as a cell pellet prior to lysis. Cells grown in cell-culture flasks should alays be trypsinized. Determine the number of cells and completely aspirate the cell-culture medium.
#Disrupt cells by adding 350uL of buffer RLT. For pelleted cells, loosen the cell pellet thoroughly by flicking the tube. Add 350uL of Buffer RLT and vortex or pipet to mix.
#To homogienize the lysate: Pipet lysate directly into a QIA shredder spin column placed in a 2 mL collection tube, and centrifuge for 2 min at full speed.
#Add 1 volume (350uL) of 70% ethanol to the homogenized lysate, and mix well by pipetting. Do not centrifuge.
#Transfer up to 700 uL of the sample, including anny precipitate that may have formed, to an RNeasy spin column placed in a 2ml collection tube. Close the lid gently, and centrifuge for 15s at >8000 x g or >10,000 rpm. Discard the flow-through.

'''Washing the Column'''
#Add 350uL Buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15s at >8000 x g or >10,000 rpm. to wash the spin column membrane. Discard the flow-through.
#Add the DNase I stock solution to 70 uL buffer RDD. Mix by gently inverting the tubem, and centrifuge briefly to collect residual liquid fro tmt the sides of the tube.
#Add the DNase I incubation mix (80uL) directly to the RNeasy spin column embrane, and place on the benchtop (20-30oC) for 15 min.
#Add 350 uL buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at >8000 x g or >10,000 rpm. Discard the clow-through.

'''Elution'''
#Add 500 uL buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at >8000 x g or >10,000 rpm. to wash the spin column membrane. This dries the spin colujmn membrane, ensuring that no ethanol is carried over during RNA elution. Residual ethanol may interfere with downstream reactions.
# Place the RNeasy spin oclumn in a new 2 ml collection tube, and discared the flowthrough. Close the lid gently, and centrifuge at full speed for 1 min.
# Place the RNeasy spin column in a new 1.5 ml collection rube. Add 30 uL RNase-free water directly to the center of the spin column membrane. Close the lid gently, and centrifuge for 1 min at >8000 x g or >10,000 rpm to elute the RNA.
# If the expected RNA yield is >30 ug, repeat the previous step using another 30-50 uL RNase-free water, or using the eluate from the previous step. Reuse the collection tube from th eprevious step.

Harvesting RNA from Cells grown in monolayer

2009-05-12T15:34:05Z

Jpecherer:

'''Loading the Column'''
#Harvest cells grown in a monolayer (do not use more than 10^7 cells): Cells can be either lysed directly in the cell-culture vessel (up to 10 cm diameter) or trypzinized and collected as a cell pellet prior to lysis. Cells grown in cell-culture flasks should alays be trypsinized. Determine the number of cells and completely aspirate the cell-culture medium.
#Disrupt cells by adding 350uL of buffer RLT. For pelleted cells, loosen the cell pellet thoroughly by flicking the tube. Add 350uL of Buffer RLT and vortex or pipet to mix.
#To homogienize the lysate: Pipet lysate directly into a QIA shredder spin column placed in a 2 mL collection tube, and centrifuge for 2 min at full speed.
#Add 1 volume (350uL) of 70% ethanol to the homogenized lysate, and mix well by pipetting. Do not centrifuge.
#Transfer up to 700 uL of the sample, including anny precipitate that may have formed, to an RNeasy spin column placed in a 2ml collection tube. Close the lid gently, and centrifuge for 15s at >8000 x g or >10,000 rpm. Discard the flow-through.

'''Washing the Column'''
#Add 350uL Buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15s at >8000 x g or >10,000 rpm. to wash the spin column membrane. Discard the flow-through.
#Add the DNase I stock solution to 70 uL buffer RDD. Mix by gently inverting the tubem, and centrifuge briefly to collect residual liquid fro tmt the sides of the tube.
#Add the DNase I incubation mix (80uL) directly to the RNeasy spin column embrane, and place on the benchtop (20-30oC) for 15 min.
#Add 350 uL buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at >8000 x g or >10,000 rpm. Discard the clow-through.

'''Elution'''
#Add 500 uL buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at >8000 x g or >10,000 rpm. to wash the spin column membrane. This dries the spin colujmn membrane, ensuring that no ethanol is carried over during RNA elution. Residual ethanol may interfere with downstream reactions.
# Place the RNeasy spin oclumn in a new 2 ml collection tube, and discared the flowthrough. Close the lid gently, and centrifuge at full speed for 1 min.
# Place the RNeasy spin column in a new 1.5 ml collection rube. Add 30 uL RNase-free water directly to the center of the spin column membrane. Close the lid gently, and centrifuge for 1 min at >8000 x g or >10,000 rpm to elute the RNA.
# If the expected RNA yield is >30 ug, repeat the previous step using another 30-50 uL RNase-free water, or using the eluate from the previous step. Reuse the collection tube from th eprevious step.

Harvesting RNA from Cells grown in monolayer

2009-05-12T15:33:35Z

Jpecherer:

'''Loading the Column'''
#Harvest cells grown in a monolayer4 (do not use more than <math>10^7</math> cells): Cells can be either lysed directly in the cell-culture vessel (up to 10 cm diameter) or trypzinized and collected as a cell pellet prior to lysis. Cells grown in cell-culture flasks should alays be trypsinized. Determine the number of cells and completely aspirate the cell-culture medium.
#Disrupt cells by adding 350uL of buffer RLT. For pelleted cells, loosen the cell pellet thoroughly by flicking the tube. Add 350uL of Buffer RLT and vortex or pipet to mix.
#To homogienize the lysate: Pipet lysate directly into a QIA shredder spin column placed in a 2 mL collection tube, and centrifuge for 2 min at full speed.
#Add 1 volume (350uL) of 70% ethanol to the homogenized lysate, and mix well by pipetting. Do not centrifuge.
#Transfer up to 700 uL of the sample, including anny precipitate that may have formed, to an RNeasy spin column placed in a 2ml collection tube. Close the lid gently, and centrifuge for 15s at >8000 x g or >10,000 rpm. Discard the flow-through.

'''Washing the Column'''
#Add 350uL Buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15s at >8000 x g or >10,000 rpm. to wash the spin column membrane. Discard the flow-through.
#Add the DNase I stock solution to 70 uL buffer RDD. Mix by gently inverting the tubem, and centrifuge briefly to collect residual liquid fro tmt the sides of the tube.
#Add the DNase I incubation mix (80uL) directly to the RNeasy spin column embrane, and place on the benchtop (20-30oC) for 15 min.
#Add 350 uL buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at >8000 x g or >10,000 rpm. Discard the clow-through.

'''Elution'''
#Add 500 uL buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at >8000 x g or >10,000 rpm. to wash the spin column membrane. This dries the spin colujmn membrane, ensuring that no ethanol is carried over during RNA elution. Residual ethanol may interfere with downstream reactions.
# Place the RNeasy spin oclumn in a new 2 ml collection tube, and discared the flowthrough. Close the lid gently, and centrifuge at full speed for 1 min.
# Place the RNeasy spin column in a new 1.5 ml collection rube. Add 30 uL RNase-free water directly to the center of the spin column membrane. Close the lid gently, and centrifuge for 1 min at >8000 x g or >10,000 rpm to elute the RNA.
# If the expected RNA yield is >30 ug, repeat the previous step using another 30-50 uL RNase-free water, or using the eluate from the previous step. Reuse the collection tube from th eprevious step.

Harvesting RNA from Cells grown in monolayer

2009-05-12T15:26:33Z

Jpecherer: Created page with ''''Loading the Column''' #Harvest cells grown in a monolayer4 (do not use more than <math>10^7</math> cells): Cells can be either lysed directly in the cell-culture vessel (up t...'

Real Time PCR From Cell Culture

2009-05-12T15:25:57Z

Jpecherer:

==Real Time qPCR==
===Materials===
*cDNA for templates
*Qiashredder and RNEasy kits from Qiagen
*Superscript Kit from Invitrogen
*SyberGreen PCR Master Mix Applied Biosystems
*96 well qPCR plate
*Primers (Dilute to 0.22 uM mixture of fwd and rev)
*Generate primers using http://pga.mgh.harvard.edu/primerbank/index.html

==Protocol==
===RNA Extraction===
#Use RNEasy kit with Qiashredder. see [[Harvesting RNA from Cells grown in monolayer]]. For a 12 well plate, use 350 uL of RLT (add 10 uL B-ME per 1 mL of RLT buffer).
#Scrape cells and pass through Qiashredder column. Do the optional DNAse step at step 5 using the RNAse free DNAse from Qiagen.
#Store at -20 until RT reaction

===RT-PCR Reaction===
#Use 8 uL of RNA per RT reaction.
#Use Superscript RT-PCR kit from Invitrogen, following manufacturers instructions
#Store cDNA at -20 until use

===Plate Preparation===
#Prepare dilutions of primers. Need 9 uL per well. Book 3h on qPCR machine
#Get 96 well block and keep on rack. Do not touch bottom of plate.
#Add 1 uL template per well.
#Add 9 uL primer per well
#Using PCR strip and multichannel pipettor, add 10 uL Master mix to each well

==References (Saltiel Lab)==
<pubmed>18829989</pubmed>
<pubmed>17008399</pubmed>
<pubmed>17200717</pubmed>
<pubmed>17192460</pubmed>
<pubmed>16926380</pubmed>

Main Page

2009-05-12T15:07:42Z

Jpecherer: /* Cell and Tissue Culture */

==Cell and Tissue Culture==
*[[Splitting Cells]]
*[[Differentiation of 3T3-L1 Cells]]
*[[Electroporation of 3T3-L1 Adipocytes]]
*[[Immunofluoresence]]
*[[3T3-L1 Adipocyte Fractionation]]
*[[Preparing Cell Lysates]]
*[[Harvesting RNA from Cells grown in monolayer]]

==Cloning and Molecular Biology==
*[[Transformation of Bacteria]]
*[[Mutagenesis]]
*[[PCR Amplification of DNA]]
*[[Restriction Enzyme Based Cloning]]
**[[Restriction Enzyme Based Cloning - Ordering Primers]]
*[[TOPO Cloning]]
*[[Preparing an Agarose Gel]]

==Protein Analysis==
*[[Western Blotting]]
*[[Surface Plasmon Resonance - Protein Lipid Interactions]]
===Protein Quantification===
*[[Bradford Assay]]
*[[Quantification by Absorbance at 280nm]]

===Protein Purification===
*[[French Press]]
*[[Purification of GST Fusion Proteins]]
*[[Determining Percent Purity]]

==Transcriptional Analysis==
*[[Real Time PCR From Cell Culture]]

==Mouse Protocols==
===Genotyping===
*[[Ear Tagging and Tail Clipping]]
*[[DNA Preparation from Tail Clip]]
*[[PCR Analysis of Tail DNA]]

Determining Percent Purity

2009-05-11T18:26:35Z

Jpecherer: Elaboration on basic protocol.

#Scan image and save as a tiff file (8-bit not 16-bit) with lanes oriented vertically.
#Open ImageJ either locally or via the webstart applet at http://rsbweb.nih.gov/ij/applets.html
#Using a rectangular box (box tool)select entire lane.
#Select lane either by selecting Analyze->Gels->Select First Lane or CTRL-1
#Plot lane by selecting Analyze->Gels->Plot Lanes or CTRL-3
#Draw baseline using the line tool.The baseline is to remove the "noise" from the blue-ness of the scanned gel. This is the reason for washing the gel with ddH20. The baseline should begin at the zero determined by where the plotted curve meets the axis.
#If necessary use the line tool to connect peaks to the baseline. Use the line tool to connect the peaks to the baseline as they would if the peaks were individual and not connected in a line. Isolate individual peaks which protrude significantly from the baseline.
#Select peaks using the wand (tracing) tool. Click on selected peaks (all peaks which are above the baseline) in order.
#Go to the results window and the areas are calculated in numerical order. Be sure to count the peaks on the plot as to determine which peak corresponds to the peak of interest.
#Copy the results into an excel file and using the sum function, sum the areas.
#Calculate purity by the area under the peak of interest divided by the area under all peaks combined.

Purification of GST Fusion Proteins

2009-05-07T15:39:36Z

Jpecherer: Updates to previous version, updatite the lysis and purification section.

==Bacteria Production and Induction==
#Express and induce protein in culture under appropriate conditions (normally innoculate the previous day)
#grow an overnight culture in ~25 mL LB/Amp (+Chloramphenicol if needed) from a colony <2 weeks post transformation
#add 5 mL culture to 1L TB/Amp and grow at 37C
#grow to OD600 of 0.6-1.0 and induce with 10 uM IPTG (optimize for each protein)
#let grow O/N at <25C (optimize induction time/temp for each protein, see [[Induction Conditions]]).
#centrifuge cells 10 min at 9000 RPM to pellet bacteria freeze if stopping at this point
==Lysis and Purification==
#Resuspend cells in ~20 mL lysis buffer (PBS + 0.1% B-ME and a PI tablet). Freeze in liquid nitrogen if not continuing with purification
#French Press cells 2 x 15 000 psi (see French Press Protocol)
#Centrifuge lysate at >15 000 RPM (i.e. 20,000RMP in JA25.5 at 4 degrees C) for 30 min to clarify
#Add 1.0 mL glutathione-agarose to 50 mL PBS (no PI) to prepare beads. Let sit for 30 min. to allow beads to settle or pellet beads for 5 min at 1000 RPM and remove buffer
#Save a lysate sample (20 uL) and add clarified lysate to equilibrated beads
#Incubate with rotation for 1h at 4C
#Pellet beads (save sample of unbound protein) and wash 3 x 50 mL cold wash buffer being careful to not suck up beads
#Pour into disposable column (BioRad # 732-6008) to collect beads. Wash with another 10 mL of PBS
#Elute with ~5 mL room temp. elution buffer, collecting 10 x ~0.5 mL fractions. Check fractions for protein with Bradford assay
*Elution buffer contains 50mM Glutathionie in PBS(0.615g in 40mL PBS), pH to between 7 and 8.
#Combine fractions with protein and dialyse O/N into 200 mL PBS in 50% glycerol (will concentrate sample ~4X)
#Measure protein concentration (Bradford or A280; concentrate if necessary and store at -20)
#Analyze uninduced and induced cells (0.5 mL culture resuspended in 100 uL 2X SDS), lysate (5 uL in 2X), unbound (5 uL in 2X) and protein (0.5-10 ug) by SDS-PAGE

French Press

2009-05-06T16:14:22Z

Jpecherer: Changes to protocol based on the non-functionality of the sample loading aparatus. More specific information on loading the cylinder into the French Press added.

==Prepare the Cell (Not Auto-Fill)==
#Pour in sample, replace base and place back on the press. Ensure valves are tightened

==Loading Sample==
#Set French Press to Down and start. Pour sample into cylinder followed by the piston.
#Ensure that the piston is lubricated with glycerol.
#Place cylinder with piston into the French Press instrument from the back, making sure that the piston's lever is orthogonal to the brace screws.
#Position the valves such that they are accessible to your hands.

==Lysing Sample==
#For bacteria 15 000 psi of cell pressure is sufficient. For a 1cm cell this corresponds to about 1100 psi on the gauge (see chart)
#Turn black knob all the way to the left
#Set Press to High and ensure both valves are securely closed
#Start the press. The cell will move until it encounters pressure and will stop moving (the motor will still be running)
#Slowly turn the knob until the gauge pressure reads desired pressure
#Slowly open valve until sample comes out, keep an eye on the pressure and make sure it doesn’t drop. Continue until piston is fully depressed. Close bottom valve
#Reload sample if necessary

==Cleaning/Storage==
#Disassemble and rinse all parts with 70% ethanol then distilled water
#Normally cells are stored at 4C to reduce sample heating

Transformation of Bacteria

2009-05-05T15:05:35Z

Jpecherer: Update

==Materials==
*Competent Cells
*Plasmid amplification use subcloning efficiency DH5a
*Cloning use OneShot TOP10 (Pink)
*Mutagenesis use XL1 Blue Supercompetent (Blue), comes in 50uL aliquots
*SOC Buffer
*DNA of interest (1 uL for plasmid, 5 uL for cloning/mutagenesis)
*Plates (Amp or Kan; in cold room)

==Protocol==
#Thaw cells on ice and label
#Add DNA to cells and mix by tapping
#Incubate on ice 30-45min
#Heat shock at 42C for 45s
#Place back on ice
#Add 450 uL of SOC Buffer or LB on shelf OR add 1000uL and adjust the level.
#Incubate at 37C for 1h with occasional mixing
#Plate 30-50 uL for amplification, or all for cloning/mutagenesis
*When plating be sure to label plates and use extreme caution when sterilizing the spreader with EtOH and fire.

Surface Plasmon Resonance - Protein Lipid Interactions

2009-05-04T20:44:48Z

Jpecherer: Additions from protocol handwritten by Dave.

==Materials==
*L1 Sensor Chip (Biacore)
*Dioleylphosphatidylcholine (Sigma) dissolved in 1:1 chloroform:methanol with 0.1% HCl
*PIPx of interest (Avanti or Echelon) dissolved in 1:1 chloroform:methanol with 0.1% HCl
*HBS-N (25mM HEPES, pH 7.4, 150 mM NaCl)(6.25mL Hepes (1M), 9.38mL NaCl (4M)) Total volume 250mL.
*1M NaOH
*pH strips
*Water bath sonicator set to 40C
*Avanti MiniExtruder
*1% beta octylglucoside
*0.5% SDS
*30% ethanol
*100mM NaOH

==Preparation of Liposomes==
#Combine DOPC + C16 lipid (or DOPC alone) at a ratio of 97:3 and dry under nitrogen (150uL PC or 145.5uL PC + 4.5 uL PI)
#Resuspend to 10 mM total lipid with HBS-N (100 uL). Vortex thoroughly and sonicate in water bath
#Correct pH to 7.4 using pH paper and 0.6uL aliquots of 1M NaOH
#Freeze thaw in a water bath sonicator at 40C and liquid nitrogen 8 times.
#Pass 10X through a polycarbonate filter using an Avanti MiniExtruder at Room Temperature.
*Rinse syringe with milliQ water.
*Wet filter paper and put the filter on the block.
*Place PC filter on block.
*Tightly attach syringe and place on blcok.
*Pass 250uL of milliQ water through the filger ~5 times.
*Discard the water and inject the sample through the filter 10 times.

==Preparation of Surface==
#Wash all four surfaces at 10 uL/min and 25C with HBS-N
#Inject 20 uL of 1% beta-octylglucoside
#Inject 20 uL of 0.5% SDS
#Inject 10 uL of 1% beta-octylglucoside
#Inject 10 uL of 30% ethanol
#Inject 70 uL of extruded lipid suspension at 10uL/min (should see an increase of 5000-10000RU) to desired well (start with lane 4 and move to lane 1-PC only, lipids can migrate so load in a way to minimize contamination affecting results)
#Wash with 20 uL of 0.1M NaOH

==Analysis of Sample==
#Inject protein samples adjusting contact time as necessary to reach saturation (typically 400s per injection)
#Inject 20 uL 0.1M NaOH between samples
#For Kd determination, inject buffer 2-3x first to get a blank reading

==Reference==
<pubmed>16829131</pubmed>