Changes

==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===1.#Use RNEasy kit with Qiashredder. For a 12 well plate, use 350 uL of RLT (add 10 uL B-ME per 1 mL of RLT buffer).2.#Scrape cells and pass through Qiashredder column. Do the optional DNAse step at step 5 using the RNAse free DNAse from Qiagen.3.#Store at -20 until RT reaction

RT-PCR Reaction===Plate Preparation===1#Prepare dilutions of primers.Use 8 Need 9 uL of RNA per RT reactionwell. Book 3h on qPCR machine 2#Get 96 well block and keep on rack. Do not touch bottom of plate.Use Superscript RT-PCR kit from Invitrogen, following manufacturers instructions3#Add 1 uL template per well.Store cDNA at -20 until use #Add 9 uL primer per well#Using PCR strip and multichannel pipettor, add 10 uL Master mix to each well

Plate Preparation1.Prepare dilutions of primers. Need 9 uL per well. Book 3h on qPCR machine 2.Get 96 well block and keep on rack. Do not touch bottom of plate.3.Add 1 uL template per well. 4.Add 9 uL primer per well5.Using PCR strip and multichannel pipettor, add 10 uL Master mix to each well ==References (Saltiel Lab)==