Real Time PCR From Cell Culture

Real Time qPCR

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

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

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)

Book 3h on qPCR machine at http://felix2.lsi.umich.edu/ORS
Prepare primer mix from 5 uM primer pair stocks by adding 0.4 uL primers + 4.6 uL water per reaction
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