---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2018-10-17 17:02:01+00:00
layout: post
slug: reverse-transcription-ronits-c-gigas-dnased-ctenidia-rna
title: Reverse Transcription - Ronit's C.gigas DNased ctenidia RNA
categories:
  - 2018
  - Miscellaneous
tags:
  - Crassostrea gigas
  - ctenidia
  - dessication
  - diploid
  - DNased RNA
  - heat shock
  - M-MLV
  - oligo dT
  - Pacific oyster
  - reverse transcriptase
  - reverse transcription
  - triploid
---

Proceeded with reverse transcription of [Ronit's DNased ctenidia RNA (from 20181016)(2018-10-16-dnase-treatment-ronits-c-gigas-ploiyddessication-ctenidia-rna.html).

Reverse transcription was performed using 100ng of each sample with M-MMLV Reverse Transcriptase from Promega.

Briefly, 100ng of DNased RNA was combined with oligo dT primers and brought up to a final volume of 15uL. Tubes were incubated for 5mins at 70oC in a PTC-200 thermal cycler (MJ Research), using a heated lid. Samples were immediately placed on ice.

A master mix of buffer, dNTPs, water, and M-MMLV reverse transcriptase was made, 10uL of the master mix was added to each sample, and mixed via finger flicking. Samples were incubated for 1hr at 42oC in a PTC-200 thermal cycler (MJ Research), using a heated lid, followed by a 5min incubation at 65oC.

Samples were stored on ice for use later this afternoon by Ronit.

Samples will be stored in Ronit's -20oC box.

Reverse transcription calcs (Google Sheet):





  * [20181017_Cgigas_ploidy_cDNA_calcs](https://docs.google.com/spreadsheets/d/12FNjWTYBOL44NqTkMhJXyAHbIA7dngZhoaswqF0aaAk/edit?usp=sharing)


