Diggestion Problems!

George Rutherford gruther at bilbo.bio.purdue.edu
Mon Mar 1 15:52:32 EST 1999


In article <36D56C17.59FD04DA at ibm.net>, Nicolas Lemee <Lemee at ibm.net> wrote:

> Are you sure Kpn I is unique ? (i.e. are you sure EcoR V is still there ?)
> 
> How close is EcoR V to Knp I ? (how close to the 'end' ?)
> Have you tried just to heat inactivate Kpn I  or do a double digest ?
> (according to the promega catalog they are both 75-100% in their
> multi-core buffer)
> Are you sure you do not carry over some phenol ?

These are points well-taken. I never, ever do an extraction as described
by the original poster. I simply do the first digest with the enzyme
requiring the lower salt buffer, adjust the salt to the level of the
higher salt requiring enzyme and digest away. I've never had a problem. As
Nicholas suggests, any phenol carryover could be causing the problem.

George



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