Reverse Transcription – Water Filter DNased RNA (from 20161207)

Performed reverse transcription on the DNased RNA samples that I verified were free of detectable RLO DNA (20161207).

Combined 17μL DNased RNA + 0.5μL random primers (Promega; Cat: C1181) in 0.2mL PCR tubes.

NOTE: The 17μL was virtually all of the sample volume recovered from DNasing. As such, the DNased RNA will not be quantified.

Incubated DNased RNA and primer mix in PTC-200 thermal cycler (MJ Research) at 70C for 5mins w/heated lid, then immediately placed on ice.

Created master mix of following components:

REAGENT SINGLE REACTION VOL (μL) NUMBER REACTIONS TOTAL VOL (μL)
5x MMLV RT BUFFER 5 14 70
10mM dNTPS (Promega) 1.25 14 17.5
MMLV RT (Promega) 0.5 14 7

Added 6.75 of master mix to each and mixed by pipetting.

Incubated PTC-200 thermal cycler (MJ Research) @ 37C for 1hr (no heated lid), followed by 95C for 3mins (heated lid). Samples were transferred to 0.5mL snap cap tubes and labelled with “cDNA” and the corresponding sample name. Samples will be stored in my -20C box.

UPDATE 20170830 Lisa has moved these samples to a -20C box dedicated to RLO Viability cDNA.

Reverse Transcription – Withering Syndrome DNased RNA

Performed reverse transcription with 307ng of each DNased withering syndrome RNA from earlier today using Promega reagents (M-MLV, random primers) according to the manufacturer’s recommendations in the M-MLV product insert.

Reverse transcription calcs are here: 20150318 – WS Viability cDNA Calcs

Incubated DNased RNA/primer mixture @ 70C for 5mins. Placed immediately on ice. Added M-MLV master mix to each sample, incubated @ 37C 1hr and held at 4C in thermalcycler.