---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2015-05-13 00:28:16+00:00
layout: post
slug: qpcr-jake-o-lurida-ctenidia-rna-control-samples-from-20150507
title: qPCR - Jake O.lurida ctenidia RNA (Control Samples) From 20150507
categories:
  - 2015
  - Miscellaneous
tags:
  - actin
  - ctenidia
  - gill
  - olympia oyster
  - Opticon2
  - Ostrea lurida
  - qPCR
  - RNA
  - 'SR ID: 1504'
  - 'SR ID: 1505'
---

Ran qPCRs on the [_O.lurida_ total RNA I isolated on 20150507](https://robertslab.github.io/sams-notebook/posts/2015/2015-05-07-rna-isolation-jakes-o-lurida-ctenidia-control-from-20150422/) to assess presence of gDNA carryover with Oly Actin primers (SR IDs: 1505, 1504).

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).

Master mix calcs are here: [20150512_qPCR_Oly_RNA](https://docs.google.com/spreadsheets/d/1-jUGGyD56GcA_uk07TFUEh2R0Y2e6DxeEzqdByTccJE/edit?usp=sharing)

Cycling params:




    
  * 95C - 3mins



40 cycles of:


    
  * 95C - 5s

    
  * 60C - 20s



Melt curve



Plate layout: [20150512_qPCR_plate_Jake_Oly_Control_RNA](https://docs.google.com/spreadsheets/d/1Ev85A7KgH3jrN6UnZd2b3gXySaX0rQqGt8_poKPcIks/edit?usp=sharing)





Results:

qPCR Data File (Opticon2): [Sam_20150512_105811.tad](https://eagle.fish.washington.edu/Arabidopsis/qPCR/Opticon/Sam_20150512_105811.tad)

qPCR Report (Google Spreadsheet): [20150512_qPCR_Report_Jake_Oly_Control_RNA](https://docs.google.com/spreadsheets/d/1F8_DalqoKNCebg54zy1Fg6OFmuwkQg90QOvi89YBJbk/edit?usp=sharing)

Excluding the no template controls (NTC), all samples produced amplification. Will require DNasing before making cDNA.

On a side note, it should be noted that the efficiencies for all of the reactions were pretty bad; probably averaging 50%. Not entirely sure why or what that indicates.

In the amplification plots below, the positive control reps are the two red lines coming up at cycle ~22.



### Amplification Plots



![](https://eagle.fish.washington.edu/Arabidopsis/20150512_qPCR_Amp_Jake_Oly_Control_RNA.JPG)(http://eagle.fish.washington.edu/Arabidopsis/20150512_qPCR_Amp_Jake_Oly_Control_RNA.JPG)







### Melt Curves



![](https://eagle.fish.washington.edu/Arabidopsis/20150512_qPCR_Melt_Jake_Oly_Control_RNA.JPG)(http://eagle.fish.washington.edu/Arabidopsis/20150512_qPCR_Melt_Jake_Oly_Control_RNA.JPG)