Currently do a lot of 16s rRNA identifications of bacteria, of which I use degenerate primers to maximise my chances of amp. I sequence with the standard Eurofins Sanger sequencing (TubSeq), but the degenerate primers are not ideal for the service and I frequently get flatline reads.
Does anyone else have this problem, or know of a better sequencing method than sending off each primer variation?
Sven Reister
Hi
One possible approach would be to use TA cloning and do sequencing with primers in the backbone. With this approach you could get a better start sequence
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Kevin Cavallin
Have you tried doubling the primer concentration for each degeneracy?
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