Shelly ordered some new primers as potential normalizing genes and asked me to test them out (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)
- 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.
Master mix calcs are here:
- 200200824_qPCR_geoduck_RPL5-v2-v3_TIF2s6b-v2-v3 (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):
CFX Data File (PCRD):
CFX Results File (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 ~75oC.
RPL5 v2
AMPLIFICATION PLOTS
MELT PLOTS
RPL5 v3
TIF3s6b v2
TIF3s6b v3
