---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
layout: post
title: qPCR - P.generosa RPL5-v2-v3 and TIF3s6b-v2-v3 Primer Tests
date: '2020-08-24 09:52'
tags:
  - qPCR
  - CFX Connect
  - Panopea generosa
  - geoduck
  - SsoFast
categories:
  - 2020
  - Miscellaneous
---
Shelly ordered some new primers as potential normalizing genes [and asked me to test them out](https://github.com/RobertsLab/resources/issues/988) (GitHub Issue).

Primers used:

| SRID | Primer_Name    |
|------|----------------|
| 1787 | RPL5_v2_FWD    |
| 1786 | RPL5_v2_REV    |
| 1785 | RPL5_v3_FWD    |
| 1784 | RPL5_v3_REV    |
| 1783 | TIF3s6b_v2_FWD |
| 1782 | TIF3s6b_v2_REV |
| 1781 | TIF3s6b_v3_FWD |
| 1780 | TIF3s6b_v3_REV |

Positive control was pooled cDNA, created by combining 2uL from each of the following:

- 11-08 1H ([made by me from 20191125](https://robertslab.github.io/sams-notebook/posts/2019/2019-11-26-Reverse-Transcription---P.generosa-DNased-Hemolypmh-and-Hemocyte-RNA-from-20191125/))
- 11-08 2H ([made by me from 20191125](https://robertslab.github.io/sams-notebook/posts/2019/2019-11-26-Reverse-Transcription---P.generosa-DNased-Hemolypmh-and-Hemocyte-RNA-from-20191125/))
- 57H ([made by me from 20191125](https://robertslab.github.io/sams-notebook/posts/2019/2019-11-26-Reverse-Transcription---P.generosa-DNased-Hemolypmh-and-Hemocyte-RNA-from-20191125/))
- 11/15 Chew (made by Kaitlyn, no date on tube)
- 11/21 Star (made by Kaitlyn, no date on tube)

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, and/or as confirmation that these primers will/not amplify gDNA.

Master mix calcs are here:

- [200200824_qPCR_geoduck_RPL5-v2-v3_TIF2s6b-v2-v3](https://docs.google.com/spreadsheets/d/1nA4_esJW8ex81fQXtI8KLdZKqDPWBFrRFoRbXU0Wfqw/edit?usp=sharing) (Google Sheet)

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


---

# RESULTS

qPCR Report (PDF):

- [sam_2020-08-24_05-17-26_BR006896.pdf](https://owl.fish.washington.edu/Athaliana/qPCR_data/qPCR_reports/sam_2020-08-24_05-17-26_BR006896.pdf)

CFX Data File (PCRD):

- [sam_2020-08-24%2005-17-26_BR006896.pcrd](https://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/sam_2020-08-24%2005-17-26_BR006896.pcrd)


CFX Results File (CSV):

- [sam_2020-08-24_05-17-26_BR006896_Quantification-Cq-Results.csv](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-24_05-17-26_BR006896_Quantification-Cq-Results.csv)



All the primers look good:

- Cq is reasonably low

- Melt curves have single peak

Amplifcation/melt plots for each primer are below.

NOTE: Genomic DNA is amplified by all three primer sets and comes up at an earlier Cq than cDNA. In `TIF3s6b v3`, gDNA exhibits a small, secondary peak at ~75<sup>o</sup>C.

##### RPL5 v2

AMPLIFICATION PLOTS

![RPL5 v2 amplification plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-24_05-17-26_BR006896_RPL5-v2_amp_plots.png)

MELT PLOTS

![RPL5 v2 melt plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-24_05-17-26_BR006896_RPL5-v2_melt_plots.png)

##### RPL5 v3

![RPL5 v3 amplification plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-24_05-17-26_BR006896_RPL5-v3_amp_plots.png)

![RPL5 v3 melt plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-24_05-17-26_BR006896_RPL5-v3_melt_plots.png)

##### TIF3s6b v2

![TIF3s6b v2 amplification plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-24_05-17-26_BR006896_TIF3s6b-v2_amp_plots.png)

![TIF3s6b v2 melt plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-24_05-17-26_BR006896_TIF3s6b-v2_melt_plots.png)


##### TIF3s6b v3

![TIF3s6b v3 amplification plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-24_05-17-26_BR006896_TIF3s6b-v3_amp_plots.png)

![TIF3s6b v3 melt plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-24_05-17-26_BR006896_TIF3s6b-v3_melt_plots.png)
