---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
layout: post
title: qPCR - C.bairdi Primer Tests on gDNA
date: '2020-02-25 13:23'
tags:
  - qPCR
  - 2x SsoFast EVA Green
  - CFX
  - SRID 1774
  - SRID 1775
  - SRID 1776
  - SRID 1777
  - SRID 1778
  - SRID 1779
  - Tanner crab
  - Chionoecetes bairdi
categories:
  - 2020
  - Tanner Crab RNAseq
---
We received the primers [I ordered on 20200220](https://robertslab.github.io/sams-notebook/posts/2020/2020-02-20-Primer-Design---C.bairdi-Primers-for-Checking-RNA-for-Residual-gDNA/) and now need to test them to see if they detect gDNA. If yes, then they're good candidates to assess the presence of residual gDNA in our RNA samples before we proceed with reverse transcription.

The three primer pairs to test are:

- SRIDs 1774/1775 (40s rRNA S30)

- SRIDS 1776/1777 (allantoicase)

- SRIDs 1778/1779 (ubiquitin thioesterase)

Used 1uL of a 1:10 dilution of the [6129_403_26 gDNA from 20200210](https://robertslab.github.io/sams-notebook/posts/2020/2020-02-10-DNA-Isolation-and-Quantification---Additional-C.bairdi-gDNA-from-Sample-6129_403_26/) as template (equates to ~4ng/uL).

All samples were run in triplicate.


qPCR Master Mix calcs (Google Sheet):

- [20200225_qPCR_cbai_gDNA_primer_tests_master_mixes](https://docs.google.com/spreadsheets/d/1zJa6J-xfW6Fv-hqTFz4B77J0hl1Pfab1r_BtphJjSOk/edit?usp=sharing)


See qPCR Report (see Results section below) for plate layout, cycling params, etc.

---

# RESULTS

qPCR data file (CFX):

- [sam_2020-02-25 2012-54-22_BR006896.pcrd](https://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/sam_2020-02-25%2012-54-22_BR006896.pcrd)

qPCR report (PDF):

- [sam_2020-02-25%2012-54-22_BR006896.pdf](https://owl.fish.washington.edu/Athaliana/qPCR_data/qPCR_reports/sam_2020-02-25%2012-54-22_BR006896.pdf)

SRIDs 1774/1775 (40s rRNA S30) and 1776/1777 (allantoicase) both amplified, while SRIDs 1778/1779 (ubiquitin thioesterase) did not. There was no amplification in any of the no template controls (NTCs).

SRIDs 1774/1775 (40s rRNA S30) generated a single peak in the melt curve, while SRIDs 1776/1777 (allantoicase) produced two peaks.

Despite the fact that SRIDs 1776/1777 (allantoicase) generated multiple peaks in the melt curve analysis, it produces a lower Cq value, so I'll go forward with this primer set for gDNA detection. Since this is just for testing for gDNA detection in our existing RNA, I'm not terribly concerned about the lack of specificity here. I would prefer to use the primer set that seems to have the higher sensitivity (i.e. produces the lowest Cq value)


---

#### Ampflication Plots

- Blue: SRIDs 1776/1777 (allantoicase)

- Green: SRIDs 1774/1775 (40s rRNA S30)

- Purple: SRIDs 1778/1779 (ubiquitin thioesterase)

 - Red: NTCs

![qPCR amplification plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-02-25%2012-54-22_amp_plots.png)



---

#### Melt Curve Plots

(same color scheme as amp plots above)


![qPCR melt curve plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-02-25%2012-54-22_melt_plots.png)