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

Bernard Murray bernard at elsie.nci.nih.gov
Fri Mar 15 15:41:38 EST 1996


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.

Aaargh!  I've been reading these posts with a degree of indifference
until this morning when I realised that a probe I've been given
recently needs to be excised with NdeI.  I guess the short half life
is inescapable but NEB make no mention about adding BSA.  Would this
stabilise the protein or does the enzyme self-destruct during catalysis?
How about extra DTT?  Also, can you compensate for dity minipreps by
adding spermidine to the digestion? - this works with other
fastidious enzymes.
	Oh for a friendly/non-illegitimate isoschizomer!

			Bernard
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov  (National Cancer Institute, NIH, Bethesda MD, USA)




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