---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2015-08-06 18:27:57+00:00
layout: post
slug: reverse-transcription-o-lurida-dnased-rna-1hr-post-mechanical-stress
title: Reverse Transcription – O.lurida DNased RNA 1hr post-mechanical stress
categories:
  - 2015
  - Olympia oyster reciprocal transplant
tags:
  - cDNA
  - ctenidia
  - DNased RNA
  - gill
  - mechanical stress
  - olympia oyster
  - Ostrea lurida
  - reverse transcriptase
  - reverse transcription
---

Performed reverse transcription on the Olympia oyster [DNased RNA from the 1hr post-mechanical stress samples](https://robertslab.github.io/sams-notebook/posts/2015/2015-07-27-dnase-treatment-o-lurida-ctenidia-1hr-post-mechanical-stress-rna/) from 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 24 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): [20150806_Jake_oly_mech_stress_cDNA_calcs](https://docs.google.com/spreadsheets/d/1bjKUhOonXp4ZEq5NSiWWQJg2hzOEE1MQ3z5_hIHAn6A/edit?usp=sharing)
