---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
layout: post
title: qPCR - Geoduck Normalizing Gene Primer Checks
date: '2020-09-14 10:22'
tags:
  - geoduck
  - Panopea generosa
  - CFX Connect
  - qPCR
categories:
  - 2020
  - Miscellaneous
---
[Shelly ordered some new primers (designed by Sam Gurr)](https://github.com/RobertsLab/resources/issues/988) (GitHub Issue) to potentially use as normalizing genes for her geoduck reproduction gene expression project and asked that I test them out.

Primers tested:

| SRID | Primer_Name |
|------|-------------|
| 1803 | 28s_v1_FWD  |
| 1802 | 28s_v1_REV  |
| 1801 | 28s_v2_FWD  |
| 1800 | 28s_v2_REV  |
| 1799 | 28s_v3_FWD  |
| 1798 | 28s_v3_REV  |
| 1797 | 28s_v4_FWD  |
| 1796 | 28s_v4_REV  |
| 1795 | EF1a_v1_FWD |
| 1794 | EF1a_v1_REV |
| 1793 | EF1a_v2_FWD |
| 1792 | EF1a_v2_REV |
| 1791 | EF1a_v3_FWD |
| 1790 | EF1a_v3_REV |
| 1789 | EF1a_v4_FWD |
| 1788 | EF1a_v4_REV |


I used pooled cDNA, created by combining 2uL from a variety of cDNA previously made by Kaitlyn (sorry, didn't document which samples contributed this time...).



I also used a 1:10 dilution of [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:

- [200200914_qPCR_geoduck_28s-v1-4_EF1a-v1-4](https://docs.google.com/spreadsheets/d/1WmQ9sJ0ANz5Z0d94BJ_2QOu1mr2XEGGN0Iol-jteTEY/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_2020-09-14_10-15-48_BR006896.pdf](https://owl.fish.washington.edu/Athaliana/qPCR_data/qPCR_reports/sam_2020-09-14_10-15-48_BR006896.pdf)

CFX Data File (PCRD):

- [sam_2020-09-14_10-15-48_BR006896.pcrd](https://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/sam_2020-09-14_10-15-48_BR006896.pcrd)


CFX Results File (CSV):

- [sam_2020-09-14_10-15-48_BR006896_Quantification-Cq-Results.csv](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-09-14_10-15-48_BR006896_Quantification-Cq-Results.csv)

Overall, all primer sets amplified gDNA and cDNA. All melt curves look really good, although for 28s I'd lean towards using 28s v4 due to the fact that the melt curve doesn't mirror the gDNA melt curve like the others do. For the same reasons, I'd lean towards using EF1a v1.

Amplifcation and melt plots for each primer set are below. Color coding:

- RED: No Template Control (NTC)

- CHARTREUSE: gDNA

- OTHER: cDNA

##### 28s v1

AMPLIFICATION PLOTS


![28s-v1 amp plots.png ](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-09-14_10-15-48_BR006896_28s-v1_amp_plots.png)

MELT PLOTS

![28s-v1 melt plots.png ](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-09-14_10-15-48_BR006896_28s-v1_melt_plots.png)

##### 28s v2

AMPLIFICATION PLOTS

![28s-v2 amp plots.png ](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-09-14_10-15-48_BR006896_28s-v2_amp_plots.png)

MELT PLOTS

![28s-v2 melt plots.png ](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-09-14_10-15-48_BR006896_28s-v2_melt_plots.png)

##### 28s v3

AMPLIFICATION PLOTS

![28s-v3 amp plots.png ](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-09-14_10-15-48_BR006896_28s-v3_amp_plots.png)

MELT PLOTS

![28s-v3 melt plots.png ](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-09-14_10-15-48_BR006896_28s-v3_melt_plots.png)

##### 28s v4

AMPLIFICATION PLOTS

![28s-v4 amp plots.png ](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-09-14_10-15-48_BR006896_28s-v4_amp_plots.png)

MELT PLOTS

![28s-v4 melt plots.png ](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-09-14_10-15-48_BR006896_28s-v4_melt_plots.png)

##### EF1a v1

AMPLIFICATION PLOTS

![EF1a-v1 amp plots.png ](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-09-14_10-15-48_BR006896_EF1a-v1_amp_plots.png)

MELT PLOTS

![EF1a-v1 melt plots.png ](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-09-14_10-15-48_BR006896_EF1a-v1_melt_plots.png)

##### EF1a v2

AMPLIFICATION PLOTS

![EF1a-v2 amp plots.png ](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-09-14_10-15-48_BR006896_EF1a-v2_amp_plots.png)

MELT PLOTS

![EF1a-v2 melt plots.png ](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-09-14_10-15-48_BR006896_EF1a-v2_melt_plots.png)

##### EF1a v3

AMPLIFICATION PLOTS

![EF1a-v3 amp plots.png ](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-09-14_10-15-48_BR006896_EF1a-v3_amp_plots.png)

MELT PLOTS

![EF1a-v3 melt plots.png ](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-09-14_10-15-48_BR006896_EF1a-v3_melt_plots.png)

##### EF1a v4

AMPLIFICATION PLOTS

![EF1a-v4 amp plots.png ](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-09-14_10-15-48_BR006896_EF1a-v4_amp_plots.png)

MELT PLOTS

![EF1a-v4 melt plots.png ](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-09-14_10-15-48_BR006896_EF1a-v4_melt_plots.png)
