I recently started by first attempt using a degenerate primer, and I haven't returned anything fruitful yet. I repeatedly get a large smear in the upper ladder but no distinct bands.
I'm using cDNA from scallop species and a touchdown 62-50 PCR protocol. I've played with concentrations of most everything at this point. 25 uL reactions made of 1 uL of primers, template, and dNTPs. 0.2 uL of platinum taq, and 2.5 uL of 10x buffer and 2.0 uL of MgCl.
I'd appreciate any tips or advice or even spit balling to come up with something else to try. Because we don't have genomes or much in the way of known sequences for these guys, degenerate primers are almost required for my work.
I have had much better doing a temperature gradient. I usually start with something like 55-65 or even 60-70 depending on the expected Tm. I use CODEHOP primers.
I suggest you design more degenerate primers and optimise those conditions
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