Used up the remainder of the one positive control gDNA that worked with all the primers in yesterday’s reaction (H.crach_h-1fg_intron, H.iris_actin_intron, H.crach_16s), so need to find a new set of gDNA to use for future positive controls. qPCR plate layout/set up is here. Anneal temp 50C. Used the following gDNA with :
06:50-9 – This was the good gDNA used as previous controls. Added 10uL of H2O to the tube in hopes of getting more useable DNA.
06:4-7 – No date/info available on tube.
07:12-15 – No date/info available on tube.
Results: Got decent signals with the H.crach_h-1fg primers for two of the three gDNAs. Will use the 07:12-15 gDNA as a positive control for tomorrow’s qPCR.
Due to lack of amplification in gDNA samples from 20090710 and 20090708 with either set of intron primers, will repeat with additional gDNA samples to make sure the primers are the problem and not the gDNA. Used the H.iris_actin_intron_Fw/Rv and the H.crach_h-1fg_intron_Fw/Rv primers. PCR setup/plate layout is here. Anneal temp 50C.
Results: Got a weak signal (C(t) ~ 37) in only the 06:50-9 rxns, but it did work with both primer sets.
Ran qPCR on gDNA (06:50-10) to test new primers (H.iris_actin_intron_Fw/Rv) designed to bind only to a region in an intron of the H.iris actin gene. Hopefully there’s enough homology between H.iris (primer source) and H.cracherodii (template source) for this to work. PCR setup/plate layout is here. Anneal temp 50C.
Ran qPCR on DNased Abalone Dg RNA (07:12 Set), gDNA (06:50-10) and clean cDNA (from 20090422) using primers (H.crach_h-1fg_intron_Fw/Rv) designed to bind only to a region in an intron of the H.cracherodii hemocyanin gene. PCR setup/plate layout is here. Anneal temp of 50C was used.
Results: No PCR products in any samples, not even the positive control. It seems that the primers don’t work. Will design new primers, probably from a different species of abalone since there was essentially only one gene in H.cracherodii that had any intron sequence available..