Today, I ran a qPCR assay with the cDNA from the desiccation + elevated temperature samples. I examined heat shock protein (HSP90) and elongation factor (EF1, normalization gene) and ran 2 duplicates for each primer. I created a mastermix with 20 µL of forward primer, 20 µL of reverse primer, 400 µL of 2x qPCR master mix, and 320 µL of DEPC-treated water. 19 µL of the mastermix was put into each well and a subsequent 1 µL of cDNA was put in for each sample (water for the negative controls and gDNA for the positive controls).
Sam confirmed that the elongation factor primer works, so we should hopefully see amplification for the EF1 primer. I’ll be in next week to check up on the data/run some more RNA extractions.
The wells are organized by rows and are in the order of: D01, D02, D09, D10, D11, D12, D19, D20, T01, T02, T09, T10, T11, T12, T19, T20 (samples cover 2 rows).
A1-A12 and B1-B4 are the EF1 replicate 1 samples.
C1-C12 and D1-D4 are the EF1 replicate 2 samples.
E1-E12 and F1-F4 are the HSP90 replicate 1 samples.
G1-G12 and H1-H4 are the HSP90 replicate 2 samples.
H5 is the no template control for EF1. H6 is the control with gDNA for EF1. and H7 is the replicate no template control for EF1. H8 is the replicate control with gDNA for EF1.
H9 is the no template control for HSP90. H10 is the control with gDNA for HSP90. H11 is the replicate no template control for HSP90. H12 is the replicate control with gDNA for HSP90.