Difference between revisions of "Real Time PCR From Cell Culture"
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===Plate Preparation=== | ===Plate Preparation=== | ||
#Book 3h on qPCR machine at http://felix2.lsi.umich.edu/ORS | #Book 3h on qPCR machine at http://felix2.lsi.umich.edu/ORS | ||
| − | #Prepare primer mix from 5 uM primer pair stocks | + | #Prepare primer mix from 5 uM primer pair stocks. Per reaction add 0.4 uL primers + 4.6 uL water. |
#Get 96 well block and keep on rack. Do not touch bottom of plate. | #Get 96 well block and keep on rack. Do not touch bottom of plate. | ||
#Add 5 uL template per well. | #Add 5 uL template per well. | ||
| − | #Add 5 uL primer per well | + | #Add 5 uL primer per well. |
#Add 10 uL per row into 8 wells of a PCR strip plus an extra 10-20 uL | #Add 10 uL per row into 8 wells of a PCR strip plus an extra 10-20 uL | ||
#Using a multichannel pipettor, add 10 uL Master mix to each well | #Using a multichannel pipettor, add 10 uL Master mix to each well | ||
Revision as of 12:09, 28 May 2009
Contents
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 5 uM mixture of fwd and rev and then make a working solution of 0.4 uL + 4.6 uL water per reaction)
- 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
- Typically dilute 5X in water (adjust for higher or lower expressing transcripts)
Plate Preparation
- Book 3h on qPCR machine at http://felix2.lsi.umich.edu/ORS
- Prepare primer mix from 5 uM primer pair stocks. Per reaction add 0.4 uL primers + 4.6 uL water.
- Get 96 well block and keep on rack. Do not touch bottom of plate.
- Add 5 uL template per well.
- Add 5 uL primer per well.
- Add 10 uL per row into 8 wells of a PCR strip plus an extra 10-20 uL
- Using a multichannel pipettor, add 10 uL Master mix to each well
- Start qPCR Machine using the machine in the Lin Lab or the Saltiel Lab machine
References (Saltiel Lab)
<pubmed>18829989</pubmed> <pubmed>17008399</pubmed> <pubmed>17200717</pubmed> <pubmed>17192460</pubmed> <pubmed>16926380</pubmed>
