Double restriction digest

Geoff Dwyer dwyer at central.murdoch.edu.au
Sat Dec 20 03:25:30 EST 1997


> 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
> >
> >

 Rick,

I would suggest that you forst restrict your plasmid with each enzyme separately (ie.
2 tubes) to ensure that both enzymes are cutting efficiently and that digestion goes
to completion.  You can then proceed with a fresh double digest if you prefer, or
alternatively, you can put one of the linearised reactions through a Wizard cleanup
column and then digest with the second enzyme.  A good method for reducing false
positives is to gel purify the linearised product.  If there is any undigested
plasmid it should migrate at a different rate and be eliminated when you cut the
fragment out of the gel.  If your insert is a PCR product the last thing you want to
do is dephosphorylate the vector.

Hope all goes well

Geoff
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