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

Join Date: Apr 2015
Posts: 234
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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 http://www.exiqon.com/lna-technology to raise the annealing temp of the custom sequencing primers.
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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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