---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
layout: post
title: qPCR - Ronit's DNased C.gigas RNA with Elongation Factor Primers
date: '2018-11-15'
tags:
  - qPCR
  - CFX Connect
  - EF1_qPCR_5'
  - EF1_qPCR_3'
  - SsoFast EvaGreen Supermix
  - DNased RNA
  - diploid
  - triploid
  -  Crassostrea gigas
  -  Pacific oyster
categories:
  - 2018
  - Miscellaneous
---
After [DNasing Ronit's RNA earlier today](https://robertslab.github.io/sams-notebook/posts/2018/2018-11-15-DNase---Ronits-C.gigas-Ctenidia-RNA/), I needed to verify the RNA was free of any contaminating gDNA.

Used elongation factor primers:

  * EF1_qPCR_5' (SRID 309)


  * EF1_qPCR_3' (SRID 310)

  [BB16 from 20090519](2009-05-15-gdna-isolation-macs-bb-and-dh-site-samples.html) was used as a positive control.

  Samples were run on Roberts Lab CFX Connect (BioRad). All samples were run in duplicate. See qPCR Report (Results section) for plate layout, cycling params, etc.

  qPCR master mix calcs (Google Sheet):

* [20181115_qPCR_Ronit_Cgigas_DNased_RNA](https://docs.google.com/spreadsheets/d/1OYVf7XhUydsK7SrhT3plEL0ykcRIrxzy1Wf7f9OOe68/edit?usp=sharing)

#### Results

Everything looks great! Nice, clean, gDNA-free RNA! Will proceed with reverse transcription.

qPCR Report (PDF):

  * [sam_2018-11-15 2013-17-42_BR006896.pdf](https://owl.fish.washington.edu/Athaliana/qPCR_data/qPCR_reports/sam_2018-11-15%2013-17-42_BR006896.pdf)

qPCR File (PCRD):

  * [sam_2018-11-15 2013-17-42_BR006896.pcrd](https://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/sam_2018-11-15%2013-17-42_BR006896.pcrd)

qPCR Data (CSV):

  * [sam_2018-11-15_13-17-42_BR006896_-__Quantification_Cq_Results.csv](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2018-11-15_13-17-42_BR006896_-__Quantification_Cq_Results.csv)


In the plots below, green is the positive control, blue are the samples, and red is the no template control (NTC).

---

##### Amplification Plots

![](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2018-11-15_2013-17-42_amp_plots.png)



* * *

##### Melt Curves

![](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2018-11-15_2013-17-42_melt_plots.png)