Double restriction digest

Dr Andrew Doherty a.doherty at bris.ac.uk
Mon Dec 15 06:01:10 EST 1997


Rick00100 wrote in message
<19971215014701.UAA22247 at ladder02.news.aol.com>...
>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

Either do the digests individually with control digests to see when
digestion has gone to completion - or to just de-phosphorylate. This will
ensure that even when cut with only one enzyme, no re-ligation will take
place. This is what I normally do and have few problems with false
positives.

Hope it helps

Andy D





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