where do you purchase your restriction enzymes?

Jered J Floyd jered at mit.edu
Tue Feb 15 21:42:11 EST 2000


Frederik Börnke <ricky_boernke at gmx.net> writes:

> > Personally, I just had a bad experience with NotI. Even at a 20-fold
> > overdigest I found it was truly pitiful at cutting supercoiled DNA. (I
> > wouldn't blame NEB for this, of course. They do mention it's bad at
> > cutting supercoiled, but they could stress it a bit more.)
> 
> We just had the same problem with NotI from Roche. How did you get
> around?

I was doing a restriction mapping so it wasn't critical that I use
NotI.  The map looked ok based on the other enzyme, and then I
sequenced it to make sure.

If you really absolutely have to use NotI, then I would try doing a
20x overdigest for at least 10 hours.  Be warned, though, that you may
see some star activity...although I'm not sure if that's as true for
supercoiled DNA.  Looks like you just can't win with NotI.

--Jered




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