Double restriction digest

Vladimir Svetlov svetlov at
Thu Dec 18 16:51:51 EST 1997

In article <19971215014701.UAA22247 at>,
rick00100 at (Rick00100) wrote:

> Hi Folks,
> I'm trying to subclone a fragment into my vector.  I cut my vector with 2
> different restriction enzymes to create sticky ends.  The problem is that
I got
> a lot of false positive transformants (after ligation to th efragment, of
> course).  I think it is because the vector was not cut completely, and I
> transformed a lot of the nicked vector to E. coli.  So my question is:  how do
> you make sure the vector is cut completely by 2 enzymes? 

You've been given a lot of good advices, of which dephosporylation is the
best bet. Another thing you might want to try - find out if anybody around
already used the vector to subclone something, so that you can cut it with
your enzymes of interest and a) by the appearance of the insert on the gel
to estimate the efficiency of digestion, b) purify the double-cut band from
the gel, which now, because of the insert size, is more easy to separate on
the gel from a relaxed and/or single-cut. This especially useful if you
cloning a series of a fragments (nested deletions etc.) flanked wityh the
same restriction sites.

Vladimir Svetlov
McArdle Lab for Cancer Research
Dept. Oncology
1400 University Ave.
Madison, WI 53706

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