NdeI: cutting problems?? anyone else?? seems silly! (fwd)

Arioch sai at topaz.microbio.uab.edu
Thu Mar 21 18:58:09 EST 1996


Bernard Murray wrote:
> 
> In article <dmorris-1503961110250001 at thermo.sbs.auckland.ac.nz>,
> dmorris at sbsnov1.auckland.ac.nz says...
> >
> >Ditto.  Use lots of enzyme, overnight incubations preferably, add more for
> >good luck every now and then, use CLEAN DNA, and allow at least six
> >base-pairs of extra sequence when designing primers containing terminal
> >NdeI sites.
> >
> >Its a bastard of an enzyme.

i dont know what your conditions are, but if possible, dont use NdeI in
double digests.  use it individually first and then follow it up with
your second enzyme (if eco or bam, then this method works cuz those 2
will cut just about anything in any condition...).  also , the amount of
glycerol in your digest is mucho importante especially if you use a
cranky enzyme like NdeI.  check the glycerol concentrations in the
buffer you use and dilute appropriately in your digest.  glycerol and
NdeI dont get along too well...cheers...
-- 
Arioch (sai at topaz.microbio.uab.edu)
Daemon prince of Chaos (a.k.a Cell and Molecular Biology Graduate
student)
The East side of the 9th plane of hell (a.k.a Dept. of Microbiology)
Dimension of Chaos (a.k.a University of Alabama at Birmingham...)



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