---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
layout: post
title: Reverse Transcription - Geoduck gonad RNA pool
date: '2018-12-06 16:08'
tags:
  - reverse transcription
  - geoduck
  - Panopea generosa
  - M-MLV
  - oligo dT
categories:
  - 2018
  - Miscellaneous
---
To be able to actually test the [vitellegenin primers I made](https://robertslab.github.io/sams-notebook/posts/2018/2018-11-29-Primer-Design---Geoduck-Vitellogenin-using-Primer3/), I needed some geoduck cDNA.

I pooled 1uL of each of the following 12 geoduck gonad RNA ([isolated previously from histology blocks](https://robertslab.github.io/sams-notebook/posts/2015/2015-05-28-bioanalyzer-geoduck-gonad-rna-quality-assessment/)):

- 02
- 04
- 07
- 09
- 38
- 41
- 46
- 51
- 65
- 67
- 68

The concentration of the pooled sample was 95ng/uL.

Used 950ng of the pooled RNA in the following reverse transcription reaction:

- 10uL RNA
- 1uL oligo dT primers (Promega)
- 4uL H<sub>2</sub>O
- Incubate 10mins @ 70<sup>o</sup>C in MJ PTC-200 (no heated lid); immediately on ice after incubation.
- 1.25uL of 10mM dNTPs (Promega)
- 5uL 5x MMLV Buffer (Promega)
- 1uL M-MLV reverse transcriptase (Promega)
- 3.75uL HH<sub>2</sub>O
- Final volume = 25uL
- Incubate 42<sup>o</sup>C in MJ PTC-200 (heated lid); 5mins @ 95<sup>o</sup>C

Will run qPCR and then stored in Sam's cDNA Box 2 in -20<sup>o</sup>C.