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AFLP low yield

AFLP low yield - Protocols and Methods Forum

AFLP low yield - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.

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Old 09-06-2006, 05:38 PM
aria johnson
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Default AFLP low yield


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
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Old 06-03-2009, 10:54 AM
Pipette Filler
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Default Re: AFLP low yield

I had a similar issue, you can try increasing template quantity by increasing the number of cycles in pre select amplification, your product yield might increases

Last edited by ibtsaranya; 06-03-2009 at 11:34 AM.
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aflp , low , yield

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