Consider the possibility that your silver stain recognizes all AFLP bands
(Eco/Eco, Eco/Mse, Mse/Mse), while your radioactive bands are labeled on
the Eco primer only [if you are running conventional AFLPs as described by
Vos et al.]. The 'monomorphic' band may be an Mse/Mse fragment that you
have never seen with radioactivity, sitting on top of the polymorphic
Eco/Mse (usually) band.
Of course I have no idea what sort of AFLP protocol you are using so this
advice may be off base.
Toby Bradshaw | (206)616-1796 (voice)
College of Forest Resources | (206)685-2692 (FAX)
University of Washington | http://poplar2.cfr.washington.edu/toby
In article <3AFFED74.4471ED9C at ncsu.edu>, Liz Parks <liz_parks at ncsu.edu> wrote:
>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
>~~~~~~~~~~~~~~~~~~~~~
>>
