Double restriction digest

Doug Pecota pecota at design.eng.uci.edu
Mon Dec 15 13:48:54 EST 1997


Rick

   I have an additional suggestion that you might try.  If your
sites are far apart and there is a unique restriction site in between
them you can cut with this enzyme in addition to the other
two.  This will greatly reduce self religation.  It worked well for me
when my two fragments were almost the same size and not easily
separated using gel electrophoresis.

Hope this helps
--------------------------------------------------------------------
Doug Pecota
University of California Irvine
pecota at eng.uci.edu

It isn't just what you know,
nor even who you know, 
but also what you know
that isn't so.
--------------------------------------------------------------------

On 15 Dec 1997, 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? 
> 
> Thanks,
> Rick
> 
> 




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