Fw: Unusual bands on silver stained AFLP

Deanne Bell dbell at qnis.net
Mon May 14 11:17:20 EST 2001


I may be on the wrong tarck and I have not done radioactive AFLPs - so
please excuse me if i am not helpful.

Did you run all of the selective amps from the same preselective product?
What is the difference between how the radioactive gel is run and how the
silver stain gel is run?

It seems to me that the radioactive label has caused a "shift / change" in
how the band runs through the gel (but I would expect to see the same
change in all the bands) Maybe there is some conformational change that
occurs in addition to the radioactive label which makes the band
polymorphic only with the conformational change.

D.Bell
----------
: From: Liz Parks <liz_parks at ncsu.edu>
: To: methods at hgmp.mrc.ac.uk
: Subject: Unusual bands on silver stained AFLP
: Date: Monday, May 14, 2001 7:36 AM
: 
: Hello all,
: I am baffled by the banding pattern on my silver stained AFLP gel.
: Normally I run radioactive AFLP, and when I find some interesting bands,
: I run a non-radioactive reaction and silver stain it so that I can cut
: out the bands and reamplify them for sequencing or cloning.  I've
: sequenced 100  bands this way, and it works really well.  However, there
: is a polymorphic band that I am interested in, that has been confirmed
: in two separate radioactive AFLP reactions, that appears to be
: monomorphic on the silver stained gel.  I repeated the PCR, and ran it a
: second time, with the same result.  There are other polymorphic bands
: that appear on the gel, so I don't think contamination is the answer.
: There is a monomorphic band below the band of interest, and the bands
: are well resolved, but I wonder if one of the denatured strands migrates
: slower with the larger band, and this was not detected with radioactive
: AFLP.  Any thoughts on what's going on, and how I can isolate my band?
: Thanks,
: Liz
: 
: --
: 
: ~~~~~~~~~~~~~~~~~~~~~
: Liz Parks
: North Carolina State University
: Department of Plant Pathology
: Raleigh, NC  27695
: ~~~~~~~~~~~~~~~~~~~~~
: 
: 

---




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