Sam’s Notebook: Reverse Transcription – P.generosa DNased Hemolypmh and Hemocyte RNA from 20191125

Performed reverse transcription on the DNased hemolymph and hemocyte RNA from yesterday.

Reverse transcription was performed using 100ng of each sample with M-MLV Reverse Transcriptase from Promega.

Briefly, 100ng of DNased RNA was combined with 1:10 dilution of oligo dT primers (Promega) and brought up to a final volume of 15uL in standard 0.5mL snap cap tubes. Tubes were incubated for 10mins at 70oC in a PTC-200 thermal cycler (MJ Research), using a heated lid. Samples were immediately placed on ice.

A master mix of buffer, dNTPs, water, and M-MLV reverse transcriptase was made, 10uL of the master mix was added to each sample, and mixed via finger flicking. Samples were incubated for 1hr at 42oC in a PTC-200 thermal cycler (MJ Research), using a heated lid, followed by a 5min incubation at 65oC.

Reverse transcription calcs (Google Sheet):

Resulting cDNA was stored in the small -20oC in:

from Sam’s Notebook