I am familiar with microsatellites, but currently trying to assess hylid DNA
using AFLP's and need advice on how to increase the yield of my 2nd amp
product, while improving the discrete bands that are barely visible in the 2
nd amp. smear.
When I run the product of the 2nd amplification ( on a 6% polyNAT gel) I get
a long (100bp - 800bp), faint smear with bands visible in the smear. Both
the lack of intensity of the product and the fact that I am not getting a
clean discrete banding pattern is unexpected because a) this is the 2nd amp.
with primers designed to increase specificity b) my positive control
(lambda) has a clear, bright, banding pattern (no smearing) . Could it be
that my annealing T is too low (not stringent enough), thereby producing
many fragments of varying sizes in addition to the ones I want? How can I
increase the overall product yield (more visible bands) while encouraging
selectivity in fragment amplification?
Loosing battle with AFLP