If you're using the adatptor sequences suggested by Vos et al (at least
the Eco/Mse ones) then you shouldn't get any Eco/Eco or MSe/Mse bands. I
used to do a lot of AFLP and would run the PCR products on an agarose or
acylamide gel, stain them with PICO green or ethidium Bromide, and cut
them out for sequencing, and only the Eco/Mse were seen on the gel. I
always design new adaptors based on the Vos sequence for this reason.
Jon
On 14 May 2001, 'Toby' H D Bradshaw wrote:
> 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
> >~~~~~~~~~~~~~~~~~~~~~
> >
> >
>>>
