qPCR - Carita Primer Test for High Resolution Melt (HRM) Curve Analysis

Ran a qPCR on Rick’s Lake Trout DNA from 4/28/2009 using primers in Carita’s CMA01 Primer Plate (Excel file). DNA was pooled (2uL from each sample), spec’d and diluted to 10ng/uL. qPCR set up is here. Plate layout matches Carita’s primer plate layout. Since we’re just looking for positive/negative samples, I ran this on the Opticon 2 despite the recent “problems” we’ve been having with it. Cycling params used with the 2x HRM M.M. are as follows:

95C - 10mins

40 cycles of:

95C - 10s

60C - 15s

72C - 25s

Results: Overall, looks good. Negative control is clean. Based on signal strength and clean, tight melting curves, the following primer sets (location in CMA01 primer/qPCR plates) will be used for HRM analysis tomorrow: C11, D3, E12, D3. Actually, never mind. Steven’s sent me contig info for the lake trout so we’ll just order and test primers that are more likely to produce good data instead of worrying about the salmonid primers.