Shelly has asked me to test some qPCR primers related to geoduck reproduction.
Table of SRIDs and primer name (sorted by primer name):
| SRID | Primer_Name |
|---|---|
| 1769 | APLP_FWD |
| 1768 | APLP_REV |
| 1761 | GSK3B_FWD |
| 1760 | GSK3B_REV |
| 1763 | NFIP1_FWD |
| 1762 | NFIP1_REV |
| 1745 | RPL5_FWD |
| 1744 | RPL5_REV |
| 1747 | SPTN1_FWD |
| 1746 | SPTN1_REV |
| 1773 | TIF3s6b_FWD |
| 1772 | TIF3s6b_REV |
Used pooled cDNA, created by combining 2uL from each of the following:
- 11-08 1H (made by me from 20191125)
- 11-08 2H (made by me from 20191125)
- 57H (made by me 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) as a potential positive control, and/or as confirmation that these primers will not amplify gDNA.
All qPCR reactions were run in duplicate. See qPCR Report (Results section below) for plate layout, cycling params, etc.
Master mix calcs are here:
- 20200723_qPCR_geoduck_primer_tests (Google Sheet)
RESULTS
qPCR Report (PDF):
CFX Data File (PCRD):
CFX Results File (CSV):
Plot color legend:
APLP: BLACK
GSK3B: CHARTREUSE
NFIP1: POWDER BLUE
RPL5: BLUE
SPTN1: LIGHT GREEN
TIF3s6B: MAGENTA
No Template Controls: RED
Amplification plots
Melt curves
No template controls (NTCs) did not generate any amplification in any of the primer sets.
All primer sets generated amplification in both cDNA and gDNA.
There are only two primer sets that produced acceptable melt curves:
APLP (BLACK melt plot)
NFIP1 (POWDER BLUE melt plot)