This is being done to check whether or not there is gDNA contamination in these cDNA and DNased RNA. Will use H.crach_h-1fg_intron primers. gDNA 07:12-15 was used as a positive control, based on results from yesterday’s qPCR. qPCR plate layout/set up is here. Anneal temp 50C.
Results: Everything came up negative, including the positive control! Also, the machine experienced an error at ~cycle 39, so no melting curve info. See below.
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..