AFLP troubles

"sally francis IACR-BB sally.francis at bbsrc.ac.uk
Thu Sep 13 10:52:59 EST 2001


Dear All,

Can anyone please help with an AFLP problem?

I have 80 samples (from plants) that were all prepared using the same DNA
extraction kit. Concentrations were assessed on an agarose gel relative to
lambda DNA and all samples were normalised, then 400ng of each digested and
ligated using the same pre-mixes. The samples were then bound to Dynal beads
and MseI-MseI ended fragments washed away before PCR. Whilst trying out
different primer combinations, I just used a subset of the 80 samples. The
problem is, the PCR step seems to be very variable. Sometimes the reactions
are really good and there is lots of product, then other times, virtually
nothing (same samples, same primers). When I use a primer combination on all
80 samples, that looked good on the small subset, some samples amplify well
and others poorly and the gel is unscorable. This effect looks random and
"good" samples are mixed in between the "bad" ones, so I don't think it's to
do with the electrophoresis. I think the PCR pre-mix is OK otherwise how
would some samples work so well? All the primers are fresh, the water is
from a newly opened tube, the enzyme etc. is in the Qiagen PCR Master Mix,
everything is set up in a laminar flow cabinet. The Mg conc was optimised on
some previous samples that worked well. We have used silver staining or 33P
and both methods give the same result. The size standard is always visible
though, so the problem is not the staining, or the radiolabelling.  

I'd be very grateful if anyone can help with this.

Best wishes,

Sally Francis 

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