Ran some qPCRs on some other primers on 20200723 and then Shelly has asked me to test some additional qPCR primers that might have acceptable melt curves and be usable as normalizing genes.

SRID	Primer_name
1771	TIF3s12_FWD
1770	TIF3s12_REV
1759	TIF3s8_FWD-1
1758	TIF3s8_REV-1
1757	TIF3s8_FWD-2
1756	TIF3s8_REV-2

NOTE: I accidentally ran the qPCR with the TIF3s8_FWD/REV-2 set, which we know is bad. So, had to perform another qPCR run with TIF3s8_FWD/REV-1. Doh!

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.

Used the same master mix calcs from 20200723:

20200723_qPCR_geoduck_primer_tests (Google Sheet)

RESULTS

qPCR Reports (PDF):

sam_2020-07-29_05-36-56_BR006896.pdf (TIF3s8_FWD/REV-2 and TIF3s12_FWD/REV)
sam_2020-07-29_06-54-35_BR00689.pdf (TIF3s8_FWD/REV-1)

CFX Data Files (PCRD):

sam_2020-07-29_05-36-56_BR006896.pcrd (TIF3s8_FWD/REV-2 and TIF3s12_FWD/REV)
sam_2020-07-29_06-54-35_BR006896.pcrd (TIF3s8_FWD/REV-1)

CFX Results Files (CSV):

sam_2020-07-29_05-36-56_BR006896-Quantification-Cq-Results.csv (TIF3s8_FWD/REV-2 and TIF3s12_FWD/REV)
sam_2020-07-29_06-54-35_BR00689_Quantification-Cq-Results.csv (TIF3s8_FWD/REV-1)

Plot color legend:

TIF3s8_FWD/REV-2: BLUE
TIF3s12_FWD/REV: GREEN
No Template Controls: RED

TIF3s8_FWD/REV-2 and TIF3s12_FWD/REV Amplification plots

TIF3s8_FWD/REV-2 (blue) and TIF3s12_FWD/REV (green) amplifcation plots

TIF3s8_FWD/REV-2 and TIF3s12_FWD/REV Melt curves

TIF3s8_FWD/REV-2 (blue) and TIF3s12_FWD/REV (green) melt curves

Plot color legend:

TIF3s8_FWD/REV-1: BLUE
No Template Controls: RED

TIF3s8_FWD/REV-1 Amplification plots

TIF3s8_FWD/REV-1 Melt curves

Alrighty, so if it’s not too confusing looking at the plots above, here’s how it breaks down, by primer set:

TIF3s8_FWD/REV-1 (bottom pair of plots): Looks great. Come up ~34 Cq and has single, narrow melt curve peak. gDNA also amplifies and produces similar results, suggesting no intron present.
TIF3s8_FWD/REV-2: Looks bad; has very broad melt curve. Not usable.
TIF3s12_FWD/REV: Looks good. Comes up ~37 Cq and has single, narrow melt curve peak. No template control seems to begin amplifying very late (>40 Cq), but produce no detectable melt curve. gDNA also amplifies and produces similar results, suggesting no intron present. Due to late relatively late amplification, it might be preferable to use TIF3s8_FWD/REV-1 as normalizing gene instead.