---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
layout: post
title: Reverse Transcription - P.generosa DNased Hemolypmh and Hemocyte RNA from 20191125
date: '2019-11-26 09:29'
tags:
  - reverse transcription
  - cDNA
  - DNased RNA
  - RNA
  - geoduck
  - Panopea generosa
  - MMLV
  - oligo dT
categories:
  - 2019
  - Miscellaneous
---
Performed reverse transcription on the [DNased hemolymph and hemocyte RNA from yesterday](https://robertslab.github.io/sams-notebook/posts/2019/2019-11-25-RNA-Isolation-and-Quantification---Geoduck-hemolymph-and-hemocyte-samples/).

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

Briefly, 100ng of DNased RNA was combined with 1:10 dilution of oligo dT primers (Promega) and brought up to a final volume of 15uL in standard 0.5mL snap cap tubes. Tubes were incubated for 10mins at 70<sup>o</sup>C 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-MLV 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 42<sup>o</sup>C in a PTC-200 thermal cycler (MJ Research), using a heated lid, followed by a 5min incubation at 65<sup>o</sup>C.

Reverse transcription calcs (Google Sheet):

- [20191126_geoduck_hemo_cDNA-calcs](https://docs.google.com/spreadsheets/d/1gzXoUYoJbOdBXwYH_KABzw88cX_YxcPCFMaHx_SC_Os/edit?usp=sharing)

Resulting cDNA was stored in the small -20<sup>o</sup>C in:

- [Sam's cDNA Box #3 A2 - A7](https://docs.google.com/spreadsheets/d/1dH1nrwITQwoYXUFSNx0-dHIjquq_Pde11pJ3Hz7K1Xc/edit#gid=0)