Shelly ordered some new primers as potential normalizing genes and asked me to test them out (GitHub Issue).
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.
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.