---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2015-05-13 00:42:48+00:00
layout: post
slug: qpcr-jake-o-lurida-ctenidia-rna-heat-shock-samples-from-20150506
title: qPCR - Jake O.lurida ctenidia RNA (Heat Shock Samples) from 20150506
categories:
  - 2015
  - Miscellaneous
tags:
  - actin
  - ctenidia
  - gill
  - heat shock
  - olympia oyster
  - Opticon2
  - Ostrea lurida
  - qPCR
  - RNA
  - 'SR ID: 1504'
  - 'SR ID: 1505'
---

Ran qPCRs on the [_O.lurida_ total RNA I isolated on 20150506](https://robertslab.github.io/sams-notebook/posts/2015/2015-05-07-rna-isolation-jakes-o-lurida-ctenidia-1hr-heat-stress-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_HS_RNA](https://docs.google.com/spreadsheets/d/1y-UxIdNQp_27qVvgZztf8pmxg_NBADD6SgPMkEKtMSI/edit?usp=sharing)



Results:

qPCR Data File (Opticon2): [Sam_20150512_123246.tad](https://eagle.fish.washington.edu/Arabidopsis/qPCR/Opticon/Sam_20150512_123246.tad)

qPCR Report (Google Spreadsheet):[20150512_qPCR_Report_Jake_Oly_HS_RNA](https://docs.google.com/spreadsheets/d/1CqfXuDfGfDf4N-T9ILctApAna-T6262TPUc1pA8KVtE/edit?usp=sharing)

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

Related to [the qPCR I ran earlier today with these same primers](https://robertslab.github.io/sams-notebook/posts/2015/2015-05-13-qpcr-jake-o-lurida-ctenidia-rna-control-samples-from-20150507/), the efficiencies of the reactions on this plate are significantly better (i.e. normal; >80% efficiencies) than the earlier qPCR. The improved efficiency would also explain why the positive control comes up two cycles earlier on this run.

In the amplification plots below, the positive control reps are the two lines coming up at cycle ~20.





### Amplification Plots



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





### Melt Curves



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