Not I question

David L. Haviland dhavilan at imm2.imm.uth.tmc.edu
Tue Apr 18 17:21:14 EST 2000


Caroline Szymeczek-Seay wrote:
> 
> Is Not I a particularly tricky enzyme?  It is behaving very
> tempermentally.  I've tested 2 different brands (Promega and Roche) with
> 2 different plasmids (1 plasmid that i purified, and one commercially
> obtained plasmid).  Interestingly, the only way I've been able to get
> Not I to cut is to use the Roche buffer with the Promega enzyme!
> 
> So is Not I just weird, or am i the only one to ever have this problem?

Caroline:

NotI is finicky.  Over the years, I've found it to work well in small
volumes (20ul) with small amounts of DNA (0.5-2ug).  Scaling up to batch
levels were dissapointing, volumes greater than 50ul with DNA in excess
of 5 ug).  For me, the DNA could not be a mere plasmid prep, or Wizzard,
but had to be CsCl prepped.

For reasons of money, we once had a Promega freezer and found their
enzymes seemed to work better with, then BMB, buffers rather than their
own.   You wouldn't be the first to make this finding!

Hope this helps,
David 
===========
David L. Haviland, Ph.D., Asst. Prof. Immunology 
University of Texas - Houston, H.S.C.
Institute of Molecular Medicine, R907
2121 W. Holcombe Blvd.,  Houston, TX  77030 
713.500.2413-Voice//713.500.2424-FAX
-----------------
If everything seems to be going so well, you have obviously 
overlooked something.
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