---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2015-05-22 22:40:27+00:00
layout: post
slug: reverse-transcription-subset-of-jakes-o-lurida-dnased-rna
title: Reverse Transcription - Subset of Jake's O.lurida DNased RNA
categories:
  - 2015
  - Miscellaneous
tags:
  - cDNA
  - ctenidia
  - DNased RNA
  - gill
  - heat shock
  - M-MLV
  - oligo dT
  - olympia oyster
  - Ostrea lurida
  - reverse transcriptase
  - reverse transcription
---

Currently don't have sufficient reagents to perform reverse transcription on the entire set of DNased RNA ([control](https://robertslab.github.io/sams-notebook/posts/2015/2015-05-14-dnase-treatment-jakes-o-lurida-ctenidia-rna-controls-from-20150507//robertslab.github.io/sams-notebook/2015/05/14/dnase-treatment-jakes-o-lurida-ctenidia-rna-1hr-heat-shock-from-20150506/) _O.lurida_ ctenidia samples). To enable Jake to start testing out some of his primers while we wait for reagents to come in, Steven suggested I generate some cDNA for him to use.

Used the following DNased RNA:




    
  * HC1

    
  * NC1

    
  * SC1

    
  * HT1 1

    
  * NT1 1

    
  * ST1 1



Reverse Transcription Calcs: [20150522_Jake_Oly_cDNA_Calcs](https://docs.google.com/spreadsheets/d/1fEZVOaSdIAv07df-BjSbeMc36--1mMfmbpIyAnHAxS8/edit?usp=sharing)

Briefly:




    
  * Reactions run in 0.5mL snap cap tubes

    
  * 250ng of DNased RNA used in each reaction

    
  * Combined DNased RNA with oligo dT primers and water; incubated 70C 5mins; immediately placed on ice

    
  * Added 6.75μL of buffer/dNTP/enzyme master mix to each sample; incubated 42C for 1hr; 95C for 3mins



Samples will be given to Jake and stored @ -20C.
