---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2012-02-09 01:51:59+00:00
layout: post
slug: qpcr-cdna-from-earlier-today
title: qPCR - cDNA from earlier today
categories:
  - 2012
  - Miscellaneous
tags:
  - 16s
  - cDNA
  - CFX96
  - Crassostrea gigas
  - EF1a
  - gDNA
  - Pacific oyster
  - qPCR
  - Vibrio tubiashii
---

Performed qPCR on all 12 samples. Used Cg_EF1aF/R2 (SR IDs: 1410 & 1412) for one set of qPCRs and Vtub_16s_F/R (SR IDs: 455 & 456) for the other set of qPCRs. Used pooled C.gigas cDNA (from [20110311](/Sam%27s+Working+Notebook+Jan+2011+-+March+2011#sjw20110311)) and RE22 DNA (provided by Elene) as positive controls for C.gigas and V.tubiashii, respectively. C.gigas gDNA (7ng of BB16 from [20110201](/Sam%27s+Working+Notebook+Jan+2011+-+March+2011#sjw20110201)) was used as a negative control for EF1a. [Master mix calcs are here](https://eagle.fish.washington.edu/Arabidopsis//Notebook%20Workup%20Files/20120208-01.jpg). Plate layout, cycling params, etc can be found in the qPCR Report (see Results). All samples were run in duplicate.

Results:

[qPCR Data File](https://eagle.fish.washington.edu/Arabidopsis/qPCR/CFX96/Roberts%20Lab_2012-02-08%2014-53-07_CC009827.pcrd) (CFX96)

[qPCR Report](https://eagle.fish.washington.edu/Arabidopsis/qPCR/CFX96/Roberts%20Lab_2012-02-08%2014-53-07_CC009827.pdf) (PDF)

C.gigas EF1a - Positive control amplified. Negative control and no template control were all clean (i.e. no amplification detected). The majority of samples had amplification, however two samples had no amplification at all (samples 132 & 136).

V.tubiashii 16s - Positive control amplified. No template controls exhibited amplification in both replicates. All samples exhibited amplifcation, however nearly all of the melt curves have multiple peaks present, suggesting that more than one target is being amplified. I suspect this is due to residual gDNA, but this fails to explain the amplification in the no template controls which also exhibited dual peaks in the melt curves.

Spoke with Steven and he suggested to skip troubleshooting the V. tubiashii 16s for now and proceed with trying to qPCR some additional V.tubiashii genes. Will talk with Elene to see if/which additional genes she has primers for.
