PstI digest failure

dbell dbell at qnis.net
Mon Jul 30 11:46:04 EST 2001


Dear Sally
I have been working with other restrxn enzymes on plant DNA and have become 
increasingly suspicious of my digests.  My latest hypothesis is that the 
high levels of methylation in plant DNA are interfering with my digests.  I 
am currently investigating isoschizomers that will cut methylated sites. 
 This may not answer your question, but I thought I would toss the idea out 
in the arena.

Deanne Bell
USDA Agricultural Research Service
2021 South Peach Avenue
Fresno, CA 93727
(559) 453-3170
(559) 453-3088 fax

On Monday, July 30, 2001 6:21 AM, "sally francis (IACR-BB)" 
[SMTP:sally.francis at bbsrc.ac.uk] 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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