qPCR - Dorothys Mussel cDNA from 20220726

Ran qPCRs on Dorothy’s mussel gill cDNA from 20220726 using the following primers:

SRID	Primer name
1821	Mg_MT10_F
1820	Mg_MT10_R
1819	Mg_MT20_F
1818	Mg_MT20_R
1817	Mg_small HSP24.1_F
1816	Mg_small HSP24.1_R
1815	Mg_SQSTM1_F
1814	Mg_SQSTM1_R
1813	Mg_HSC70_F
1812	Mg_HSC70_R
1811	Mg_HSP90_F
1810	Mg_HSP90_R
1809	Mg_ferritin_F
1808	Mg_ferritin_R
1807	Mg_GADD45 gamma_F
1806	Mg_GADD45 gamma_R

Mg_GADD45_gamma_F/R primers were run in triplicate. After discussion with Steven, it was determined that duplicates would be sufficient for the time being, so all other primers were run in duplicate.

All reactions were run with 2x Sso Fast EVAgreen Master Mix (BioRad) using with the following master mix calcs (Google Sheet):

20220727 - qPCR Calcs Dorothy Mussel Gill cDNA

All reactions were run in white, low profile PCR plates, using optically clear adhesive film on a CFX Connect thermal cycler (BioRad). See RESULTS below for cycling params.

RESULTS

Mg_ferritin did not produce any amplification. Will likely re-run just to ensure there wasn’t an error when creating the master mix (i.e. forgot to add primer(s)?).

The plate with Mg_HSC70 and Mg_small HSP24.1 should be reviewed to examine melt curves, as it appears there is some non-specific amplification occurring.

Additionally, it appears that Mg_HSC70 is expressed highly and stably. As such, this might be a good candidate for a normalizing gene despite our interest in its response to heat stress.