---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2015-08-06 13:51:59+00:00
layout: post
slug: qpcr-jakes-o-lurida-ctenidia-1hr-post-mechanical-stress-dnased-rna
title: qPCR - Jake's O.lurida ctenidia 1hr post-mechanical stress DNased RNA
categories:
  - 2015
  - Olympia oyster reciprocal transplant
tags:
  - actin
  - ctenidia
  - DNased RNA
  - gill
  - mechanical stress
  - olympia oyster
  - Opticon2
  - Ostrea lurida
  - qPCR
  - 'SR ID: 1504'
  - 'SR ID: 1505'
  - SsoFast EvaGreen Supermix
---

Ran qPCR on DNased RNA from earlier today to assess whether there was any residual gDNA after [the DNase treatment](https://robertslab.github.io/sams-notebook/posts/2015/2015-05-14-dnase-treatment-jakes-o-lurida-ctenidia-rna-controls-from-20150507/) with Oly_Actin_F/R 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).

Cycling params:





  * 95C – 2.5mins


  * 40 cycles of:


    * 95C – 10s


    * 60C – 20s





  * Melt curve



Master mix calcs are here: [201500806_qPCR_Oly_DNased_RNA](https://docs.google.com/spreadsheets/d/1m-rsj3HRVztBFhIBsNaB95QoRT1XKY0frtplN2jhYL0/edit?usp=sharing)

qPCR Plate Layout: [20150806_qPCR_plate_Jake_Oly_DNased_RNA](https://docs.google.com/spreadsheets/d/1m0Iz6TNFUOQsDPKaPUjn5Ap2lQRlemajBg8QlWkiVp4/edit?usp=sharing)

---


RESULTS:

qPCR Data File (Opticon):

- [20150806_165044.tad](https://eagle.fish.washington.edu/Arabidopsis/qPCR/Opticon/20150806_165044.tad)

qPCR Report (Google Spreadsheet):
- [20150806_qPCR_Report_Jake_Oly_DNased_RNA](https://docs.google.com/spreadsheets/d/1Dx4Xmc2DAqd2UBoh_7dSAfmLK0Sk6pc1K8XNVxzJoFM/edit?usp=sharing)

Positive control comes up around cycle ~21.

No amplification in the no template controls.

Two wells of the DNased RNA samples exhibit amplification (E3, F6), however the corresponding respective replicate does not. Will proceed with reverse transcription.





### Amplification Plots



Positive Controls

![](https://eagle.fish.washington.edu/Arabidopsis/20150806_qPCR_Oly_mech_stress_DNAsedRNA_PosControlsAmp.JPG)



---

### Melt Curves





#### Positive Controls (HL1)





#### ![](https://eagle.fish.washington.edu/Arabidopsis/20150806_qPCR_Oly_mech_stress_DNAsedRNA_PosControlsMelt.JPG)


---

#### DNased RNA Samples



Follow the green and red lines with the vertical bars. The different colors reflect that those are two different samples. Additionally, their respective replicates do not exhibit amplification.

![](https://eagle.fish.washington.edu/Arabidopsis/20150806_qPCR_Oly_mech_stress_DNAsedRNA_Melt.JPG)
