Double restriction digest
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
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
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