---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
layout: post
title: Reverse Transcription - Ronit's C.gigas DNased RNA from 20181115
date: '2018-11-20'
tags:
  - reverse transcription
  - cDNA
  - MMLV
  - pacific oyster
  - Crassostrea gigas
  - diploid
  - triploid
  - dessication
categories:
  - 2018
  - Miscellaneous
---
Quantified Ronit's DNased RNA [earlier today](https://robertslab.github.io/sams-notebook/posts/2018/2018-11-20-RNA-Quantification---Ronits-C.gigas-DNased-RNA-from-20181115/) and proceeded with reverse transcription using 100ng of DNased RNA with oligo dT primers (Promega) and M-MLV reverse transcriptase (Promega) according to the manufacturer's protocol.

RNA and oligo dTs were incubated at 70<sup>o</sup>C for 10mins in a PTC-200 thermal cycler (MJ Research) without a heated lid and immediately placed on ice.

A master mix of buffer, dNTPs, and M-MLV was distributed to each sample (10uL to each sample), mixed, and incubated at 42<sup>o</sup>C for 1hr, 3min at 95<sup>o</sup>C, and then held overnight at 4<sup>o</sup>C in the PTC-200 thermal cycler.

A 1:5 working dilution of each cDNA (5uL cDNA + 20uL PCR H<sub>2</sub>O was made and will be used for all subsequent qPCRs.

All samples were stored in Ronit's -20<sup>o</sup>C box.

Reverse transcription calcs (Google Sheet):

- [201811120_Cgigas_ploidy_cDNA_calcs](https://docs.google.com/spreadsheets/d/1PtFerDO6iTCrimMAoaYlEUtb7d9PbGQxtIJoRQK2CnM/edit?usp=sharing)

Info was added to Ronit's master sheet (Google Sheet):

- [Exposure 8/29-8/30 C.Gigas](https://docs.google.com/spreadsheets/d/17mv8gMbmaldggA8Zf0RwBeNF_O4faY8dJFg31XO63K4/edit?usp=sharing)