PstI digest failure

Jim Kami jakami at ucdavis.edu
Tue Aug 21 20:25:04 EST 2001


Hi,
    We've also had a lot of problems cutting plant DNA (Phaseolus sp.) with
Pst1 for the simple reason that it is methylation sensitive. Check out the New
England Biolabs site [ www.neb.com ] look under Pst1 and check out the "more
information" link.
We actually did have better success finding RFLPs with Pst1 than with other
enzymes. The general thought is that transcriptionally active regions would be
de-methylated and thus more susceptible to Pst1 than non-transcribed (and
possibly highly repetitive) DNA. Unfortunately, while there are other enzymes
with the same recognition site (XmaII, Xpa I for example) none are currently
available at this time.
Hope this helps

Jim Kami
Dept. of Agronomy and Range Science
University of California
Davis, California  95616
Ph. 1-530-752-9982
FAX  1-530-752-4361
email:  jakami at ucdavis.edu


"sally francis (IACR-BB)" wrote:

> Dear All,
>
> Has anyone ever had problems cutting plant genomic DNA with PstI? The DNA
> looks OK when run on a gel, and it can be cut with other enzymes (e.g.
> EcoRI) OK. I have tried using a massive amount of PstI (20U) to cut only
> 400ng in a 50ul reaction, but it still doesn't cut. I tried long reaction
> times (24h) - no success. Other people's batches of enzyme from the same
> manufacturer (NEB) won't work and neither will PstI from another
> manufacturer (Promega). My own PstI cuts plasmids OK, by the way. I can't
> use another enzyme for my work, so any helpful suggestions would be greatly
> appreciated!
>
> Thanks,
>
> S. Francis
>
> ---




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