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Old 03-21-2017, 08:30 AM   #6
Senior Member
Location: CT

Join Date: Apr 2015
Posts: 234

check the tm of your primers. The reverse emp primer is too cool (62-65, should be min 65). If your degeneracies are even cooler you will likely not get good annealing. I've had decent luck with locking nucleotide sequencing primers from to raise the annealing temp of the custom sequencing primers.
Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.
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