Changes

#Use Book PCR Machine for 2h#Thaw, mix and quickly spin RNA, dNTP mix, random hexamers, 10X RT buffer, 25 mM MgCl2 water#Combine the following:##8 uL of RNA per ##1 uL dNTP mix##1 uL Random Hexamers#Put in PCR machine and run program '''RT reaction.65C''' (takes 5 min)#Use Superscript In a separate tube add in the following order (for 10 reactions):##20 uL 10X RT-PCR kit buffer##40 uL 25 mM MgCl2##20 uL 0.1M DTT##10 uL RNAse Out#Add 9 uL of the reaction mix to each primer/RNA mixture from Invitrogenprevious step, following manufacturers instructionsmix and quickly centrifuge#Sit on the bench for 2 min#Add 1 uL SuperScript II RT to each tube#Put in PCR machine and run program '''RT Reaction'''. After run (75 min) place on ice.#Add 1 uL RNase H and put in PCR machine and run program '''RT 37'''. Takes 20 min.#Store cDNA at -20 until use#Typically dilute 5X in water (adjust for higher or lower expressing transcripts).