---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2015-06-17 21:13:46+00:00
layout: post
slug: reverse-transcription-o-lurida-dnased-rna-controls-and-1hr-heat-shock
title: Reverse Transcription - O.lurida DNased RNA Controls and 1hr Heat Shock
categories:
  - 2015
  - Miscellaneous
tags:
  - DNased RNA
  - M-MLV
  - oligo dT
  - olympia oyster
  - Ostrea lurida
  - reverse transcription
---

Performed reverse transcription on the Olympia oyster DNased RNA from [the control samples](https://robertslab.github.io/sams-notebook/posts/2015/2015-05-14-dnase-treatment-jakes-o-lurida-ctenidia-rna-controls-from-20150507/) and [the 1hr heat shock samples](https://robertslab.github.io/sams-notebook/2015/05/14/dnase-treatment-jakes-o-lurida-ctenidia-rna-1hr-heat-shock-from-20150506/) of Jake's project. To accommodate the large numbers of anticipated genes to be targeted in subsequent qPCRs, I prepared 100μL reactions (normally, 25μL reactions are prepared) using 250ng of each DNased RNA. A 1:10 dilution of the oligo dT primers (Promega) was prepared to improve pipetting accuracy. All incubations were performed in a thermal cycler without using a heated lid.

DNased RNA was combined with NanoPure H2O and oligo dT primers in 48 wells of a PCR plate, heated @ 70C for 10mins and immediately placed on ice. After 5mins, the plate was spun 2000g @ RT for 2mins and returned to ice.

25.25μL of a master mix containing 5x M-MLV Buffer (Promega), dNTPs (10mM each; Promega), and M-MLV Reverse Transcriptase (50U/rxn; Promega) was distributed to each well and mixed via pipetting. The plate was heated @ 42C for 1hr, 95C for 3mins. The plate was spun 2000g @ RT for 2mins and then stored @ -20C.

Plate layout and all calculations can be found here (Google Sheet): [20150616_Jake_Oly_cDNA_Calcs](https://docs.google.com/spreadsheets/d/11cB6J1wahOq4jPk6DWENAnUweyEAeHT25Zbba1RXsks/edit?usp=sharing)