Quantified Ronit’s DNased RNA earlier today and proceeded with reverse transcription using 500ng of DNased RNA with oligo dT primers (Promega) and M-MLV reverse transcriptase (Promega) according to the manufacturer’s protocol.
RNA and oligo dTs were incubated at 70oC for 10mins in a PTC-200 thermal cycler (MJ Research) without a heated lid and immediately placed on ice.
A master mix of buffer, dNTPs, and M-MLV was distributed to each sample (10uL to each sample), mixed, and incubated at 42oC for 1hr, 3min at 95oC, and then held overnight at 4oC in the PTC-200 thermal cycler.
A 1:5 working dilution of each cDNA (5uL cDNA + 20uL PCR H2O was made and will be used for all subsequent qPCRs.
All samples were stored in Ronit’s -20oC box.
Reverse transcription calcs (Google Sheet):
Info was added to Ronit’s master sheet (Google Sheet):