restriction enzymes for AFLP
Mr JN Goulding
jgouldin at hgmp.mrc.ac.uk
Thu Oct 5 03:59:33 EST 2000
On Fri, 29 Sep 2000, Dr. Duncan Clark wrote:
> In article <8r26au$e$1 at snipp.uninett.no>, Thomas Moen
> <thomas.moen at akvaforsk.nlh.no> writes
> >2) Seems to me it would be a good idea to pick enzymes that are not
> >methylation-sensitive. Then why is PstI one of the the most frequently used
> >6-cutters after EcoRI ?
> 'Cos Pst I is not sensitive to methylation.
> From what little I know about AFLP you initially use a four cutter
> enzyme and the six cutter enzyme must ligate to that four cutter enzyme
Doesn't matter which enzymes you use- the adaptors are designed to match
the restiction site of the indivdual enzyme. You don't need matching
sites. In fact, you don't want the sites to match, as the adaptors are
designed not to amplify the Eco-eco or the mse-mse fragments.
> With EcoR I that is easy but for Pst I, from memory, there is no four
> cutter with the appropriate overhang. So I would choose your four cutter
> first, as there really are only a few with overhangs and no methylation
> sensitivity, and then match up the six cutter RE.
> The problem with being on the cutting edge is that you occasionally get
> sliced from time to time....
> Duncan Clark
> DNAmp Ltd.
> Tel: +44(0)1252376288
> FAX: +44(0)8701640382
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