---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
layout: post
title: qPCR - Geoduck gonad cDNA with vitellogenin primers
date: '2018-12-06 16:10'
tags:
  - qPCR
  - CFX
  - 2x SsoFast EVAgreen
  - geoduck
  - vtg
  - vitellogenin
  - Panopea generosa
  - gonad
categories:
  - 2018
  - Miscellaneous
---
[Earlier today I made some cDNA from geoduck gonad RNA](https://robertslab.github.io/sams-notebook/posts/2018/2018-12-06-Reverse-Transcription-Geoduck-gonad-RNA-pool/) for use in this qPCR to test out the [vitellogenin primers I designed on 20181129](https://robertslab.github.io/sams-notebook/2018/11/29/Primer-Design-Geoduck-Vitellogenin-using-Primer3/)

I also used [geoduck gDNA (162ng/uL; from 20170105)](https://robertslab.github.io/sams-notebook/posts/2017/2017-01-05-dna-isolation-geoduck-gdna-for-illumina-initiated-sequencing-project/) as a potential positive control, or as confirmation that these primers will _not_ amplify gDNA.

Primers:

[SR IDs](https://docs.google.com/spreadsheets/d/14m2kkFhxcoKWWIGoAD_7VOVsAg9wilME2UcSLqfnqLI/edit?usp=sharing&authkey=CP736rQK):

- 1712
- 1711

All qPCR reactions were run in duplicate. See qPCR Report (Results section below) for plate layout, cycling params, etc.

qPCR Master Mix calcs (Google Sheet):

- [20181206_qPCR_geoduck_vtg_primer_test](https://docs.google.com/spreadsheets/d/1xhNOLrTybRBcK-kXmFfpXkq1kCTxmJVB7OnvSaQ4vfI/edit?usp=sharing)

---

#### Results

qPCR Report (PDF):

- [sam_2018-12-06 2013-24-33_BR006896.pdf](http://owl.fish.washington.edu/Athaliana/qPCR_data/qPCR_reports/sam_2018-12-06%2013-24-33_BR006896.pdf)

CFX Data File (PCRD):

- [sam_2018-12-06 2013-24-33_BR006896.pcrd](http://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/sam_2018-12-06%2013-24-33_BR006896.pcrd)

Primers worked perfectly (although, expression comes up a bit later than I'd like)! Good amplification, single peak in melt curve, and no amplification in gDNA.

This also means I can use this cDNA as a positive control for future geoduck VTG qPCRs.

---

Key for plots below:

- Green = cDNA
- Blue = gDNA
- Red = No Template Controls (NTC)

###### AMPLIFCATION PLOT

![](http://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2018-12-06_13-24-33_amp_plots.png)

---

###### MELT CURVE

![](http://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2018-12-06_13-24-33_melt_plots.png)