---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2015-05-22 00:38:41+00:00
layout: post
slug: qpcr-re-run-jakes-o-lurida-dnased-rna-samples-nc1-sc1-sc2-sc4-from-20150514
title: qPCR - Re-run Jake's O.lurida DNased RNA Samples NC1, SC1, SC2, SC4 from 20150514
categories:
  - 2015
  - Miscellaneous
tags:
  - actin
  - DNased RNA
  - olympia oyster
  - Opticon
  - Opticon2
  - Ostrea lurida
  - qPCR
  - 'SR ID: 1504'
  - 'SR ID: 1505'
  - SsoFast EvaGreen Supermix
---

The following DNased RNA samples [showed inconsistencies between qPCR reps (one rep showed amplification, the other rep did not) on 20150514](https://robertslab.github.io/sams-notebook/posts/2015/2015-05-15-qpcr-jakes-o-lurida-ctenidia-dnased-rna-control-samples/):




    
  * NC1

    
  * SC1

    
  * SC2

    
  * SC4



Reran these four samples to obtain a definitive answer as to whether or not they have residual gDNA in them prior to using them to make cDNA.

Used Oly_Actin primers (SR IDs: 1504, 1505)

Used 1μL from all templates.

All samples were run in duplicate.

Positive control was [HL1 _O.lurida_ DNA isolated by Jake on 20150323](https://heareresearch.blogspot.com/2015/03/3-23-2015-ezna-dna-isolation-with-seed.html).

Cycling params:




    
  * 95C – 2.5mins

    
  * 40 cycles of:

    
    * 95C – 10s

    
    * 60C – 20s




    
  * Melt curve



Master mix calcs: [20150521_qPCR_Oly_DNased_RNA](https://docs.google.com/spreadsheets/d/1AVf-RgRTnR65N62REZ8RWKKucuh6IAlZpuaiUbs5uOY/edit?usp=sharing)

Plate layout: [20150521_qPCR_plate_Jake_Oly_DNased_RNA](https://docs.google.com/spreadsheets/d/1yijvNDdUAPcy-HhrkHjr1R3LwdFCQTj5ci02CraQa2k/edit?usp=sharing)



Results:

qPCR Data File (Opticon): [Sam_20150521_145749.tad](https://eagle.fish.washington.edu/Arabidopsis/qPCR/Opticon/Sam_20150521_145749.tad)
qPCR Report (Google Sheet): [20150521_qPCR_Report_Jake_Oly_DNased_RNA](https://docs.google.com/spreadsheets/d/1ey-Cih6uJxoUpskVAGJS_DfO3Zc3SJzvpcPTno6qNQA/edit?usp=sharing)



No amplification in any of the RNA samples, nor the NTCs. Will make cDNA.





### Amplification Plots



![](https://eagle.fish.washington.edu/Arabidopsis/20150521_qPCR_Amp_Jake_Oly_DNased%20RNA_.JPG)(http://eagle.fish.washington.edu/Arabidopsis/20150521_qPCR_Amp_Jake_Oly_DNased%20RNA_.JPG)







### Melt Curves



![](https://eagle.fish.washington.edu/Arabidopsis/20150521_qPCR_Melt_Jake_Oly_DNased%20RNA_.JPG)(http://eagle.fish.washington.edu/Arabidopsis/20150521_qPCR_Melt_Jake_Oly_DNased%20RNA_.JPG)