Fw: Fw: Unusual bands on silver stained AFLP

Deanne Bell dbell at qnis.net
Fri May 25 16:43:37 EST 2001


Hey Liz
Are you still out there?
How's it going with the "FLOPpy"  AFLP??

D.  Bell

----------
: From: Liz Parks <liz_parks at ncsu.edu>
: To: methods at hgmp.mrc.ac.uk
: Subject: Re: Fw: Unusual bands on silver stained AFLP
: Date: Friday, May 18, 2001 11:53 AM
: 
: Actually, I think the term "preferential amplification" is more
appropriate.  This
: is discussed in the original AFLP paper by Vos.  The Eco-Mse fragments
are
: preferentially amplified over the Mse-Mse fragments, and the Eco-Eco
fragments are
: rare and too large to be efficiently amplified.  It makes sense that you
would see
: amplification with only the Eco primer--your template is many short
fragments,
: which can be efficiently amplified unidirectionally.
: 
: With that said, it looks likely that I have stumbled across one of these
rare
: cases, of course, with a very important fragment that I really want to
sequence.  I
: ran out the radioactive PCR again, and reconfirmed the correct pattern. 
I then
: silver stained these same fragments, and the pattern was different from
the
: radioactive.  I was thinking about doing asymmetrical PCR on the cut-out
fragment
: on different samples, some that are supposed to have the band, and some
that are
: not, and see what happens.
: I'll let you know what happens!
: Liz
: 
: Tim Jackson wrote:
: 
: > i'm not sure i agree with you, deanne.
: > i don't think there is any means for the pre-selective amp to
"specifically
: > amplify" Eco/Mse fragments.  there is no control in either PCR to
prevent
: > amplification of Mse/Mse fragments or Eco/Eco fragments (i've tried
throwing
: > only Eco primers in the selective amp - i thought something was wrong
with my
: > Mse primers so wanted a control - and saw tons of product on an agarose
gel!).
: > so, i think toby might be onto something.
: > perhaps you want to try amplifying what you cut out of your silver
stained gel
: > in an asymetrical PCR (normal or higher [Eco-NNN] and very low
[Mse-NNN]).  or
: > maybe you could skip the Mse primer altogether.  that will give you
single
: > stranded product but should certainly tell you it's an Mse-Mse fragment
causing
: > you problems.
: >
: > good luck!
: >
: > tim
: >
: > Deanne Bell wrote:
: >
: > > Hi Toby
: > >
: > > This is a nice thought, but the AFLP process goes through 2
amplifications:
: > > a)  the pre-selective amplification sorts out all of the Eco/Eco &
Mse/Mse
: > > fragments because it specifically amplifies the Eco/Mse fragments
: > > b)  the second amplification (selective amp) "selects" sub-sets of
those
: > > Eco/Mse fragments by extending the primers by 1 or 2 bases.
: > >
: > > By the time the product is run out on a gel, I don't think any
residual
: > > Eco/Eco or Mse/Mse fragments would be detectable.
: > > D.Bell
: > >
: > > ----------
: > > : From: 'Toby' H D Bradshaw <toby at u.washington.edu>
: > > : To: gen-link at hgmp.mrc.ac.uk.methods@hgmp.mrc.ac.uk
: > > : Subject: Re: Unusual bands on silver stained AFLP
: > > : Date: Monday, May 14, 2001 11:51 AM
: > > :
: > > : 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
: > > : >~~~~~~~~~~~~~~~~~~~~~
: > > : >
: > > : >
: > > :
: > > :
: > >
: > > ---
: 
: --
: 
: ~~~~~~~~~~~~~~~~~~~~~
: Liz Parks
: North Carolina State University
: Department of Plant Pathology
: Raleigh, NC  27695
: ~~~~~~~~~~~~~~~~~~~~~
: 
: 

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