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)

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RESULTS

qPCR Report (PDF):

• sam_2020-07-23_09-33-48_BR006896.pdf

CFX Data File (PCRD):

• sam_2020-07-23%2009-33-48_BR006896.pcrd

CFX Results File (CSV):

• sam_2020-07-23_09-33-48_BR006896-Quantification-Cq-Results.csv

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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

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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)