digestion of PCR reaction

Wolfgang Schechinger hubahopp at gmx.de
Fri Jul 23 18:17:44 EST 2004


Dear ?
it is very likely that you are right. Basically, the experiment is simple:
divide your PCR reaction and treat one with a gel filtration febore you add
the REs. Then ligate and watch out what will happen. 

But as even traces of polymerases tend to drive paranoid people crazy by
surviving gel filtration colums, you might kill your polymerases by adding
a little phenol to your pcr rxn before you begin the cleanup.

Best regards,

Wolfgang
 


At 15:04 21.07.2004 +0200, you wrote:
>Dear NG
>I have a basic question.
>Is it possible to restriction digest a PCR product directly from the
>reaction mix containing a proofreading enzyme. I am afraid that the enzyme
>(called phusion) during the digest will remove the protruding ends so I
>can't clone it directly into a cut vector?
>Usually I PCR purify it on a column, I just wondered whether this step was
>necessary. Thanks
>
>
>
Wolfgang Schechinger


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