---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
layout: post
title: qPCR - Geoduck hemolymph and hemocyte cDNA with vitellogenin primers
date: '2019-11-27 10:22'
tags:
  - qPCR:SsoFast EvaGreen
  - Panopea generosa
  - geoduck
  - vitellogenin
categories:
  - 2019
  - Miscellaneous
---
Previously [isolated RNA on 20191125](https://robertslab.github.io/sams-notebook/posts/2019/2019-11-25-RNA-Isolation-and-Quantification-Geoduck-hemolymph-and-hemocyte-samples/) and [made cDNA on 20191126](https://robertslab.github.io/sams-notebook/2019/11/26/Reverse-Transcription-P.generosa-DNased-Hemolypmh-and-Hemocyte-RNA-from-20191125/) from some geoduck hemolymph and hemocyte samples that [Shelly asked me to run qPCRs on](https://github.com/RobertsLab/resources/issues/763).

Ran qPCR with vitellogenin primers I [previously designed on 20181129](https://robertslab.github.io/sams-notebook/posts/2018/2018-11-29-Primer-Design-Geoduck-Vitellogenin-using-Primer3/) and [tested on 20181206](https://robertslab.github.io/sams-notebook/2018/12/06/qPCR-Geoduck-gonad-cDNA-with-vitellogenin-primers/)

Primers:

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

- 1712 (Pg_DN51983i8_1471)
- 1711 (Pg_DN51983i8_1347)

Positive control used was same [pooled gonad cDNA used succesffully on 20181206 to test the assay](https://robertslab.github.io/sams-notebook/posts/2018/2018-12-06-qPCR---Geoduck-gonad-cDNA-with-vitellogenin-primers/). Due to sample limitations, positive control was _not_ run in duplicate!

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):

- [20191127_qPCR_geoduck_vtg_hemo](https://docs.google.com/spreadsheets/d/16kMsHtiXdc94nVEcCczZBqqogyEsJ-qvm0jwOp55JFU/edit?usp=sharing)

NOTE: I actually ended up running this twice (55<sup>o</sup>C and 60<sup>o</sup>C)due to poor melt curves when run at 55<sup>o</sup>C anneal.

---

#### Results

qPCR at 55<sup>o</sup>C anneal:

qPCR Report (PDF):

- [sam_2019-11-27 2007-49-21_BR006896.pdf](https://owl.fish.washington.edu/Athaliana/qPCR_data/qPCR_reports/sam_2019-11-27%2007-49-21_BR006896.pdf)

CFX Data File (PCRD):

- [sam_2019-11-27%2007-49-21_BR006896.pcrd](https://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/sam_2019-11-27%2007-49-21_BR006896.pcrd)


CFX Results File (CSV):

- [sam_2019-11-27_07-49-21_BR006896_-_20_Quantification_Cq_Results.csv](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2019-11-27_07-49-21_BR006896_-%20_Quantification_Cq_Results.csv)


#### Amplifcation plots

![Amplifcation plots of VTG cDNA at 55C annealing temp](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2019-11-27_07-49-21_amp_plots.png)

#### Melt curves
![Melt curves of VTG cDNA at 55C annealing temp](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2019-11-27_07-49-21_melt_plots.png)


OK, here's the quick rundown:

- No amplification in 39H

- Single melt peaks in 31H, 61H, and positive control only

Seeing this, I decided to run this at a higher annealing temp to see if I could increase specificity (see below).



---

qPCR at 60<sup>o</sup>C anneal:

qPCR Report (PDF):

- [sam_2019-11-27_10-02-00_BR006896.pdf](https://owl.fish.washington.edu/Athaliana/qPCR_data/qPCR_reports/sam_2019-11-27_10-02-00_BR006896.pdf)

CFX Data File (PCRD):

- [sam_2019-11-27%2010-02-00_BR006896.pcrd](https://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/sam_2019-11-27%2010-02-00_BR006896.pcrd)

CFX Results File (CSV):

- [sam_2019-11-27_10-02-00_BR006896_-__Quantification_Cq_Results.csv](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2019-11-27_10-02-00_BR006896_-__Quantification_Cq_Results.csv)


#### Amplification plots

![Amplifcation plots of VTG cDNA at 60C annealing temp](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2019-11-27_10-02-00_amp_plots.png)

#### Melt curves

![Melt curves of VTG cDNA at 60C annealing temp](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2019-11-27_10-02-00_melt_plots.png)


OK, here's the quick rundown:

- Still no amplification in 39H

- All samples show single melt peak now
