if two estriction sites too close
John R. McQuiston
zje8 at cdc.gov
Fri Feb 5 07:30:32 EST 1999
I've used this method with a antibiotic resistance gene. It works really well
if you put the marker gene in one site, then cut with the other enzyme first.
Then after a couple of hours add the second enzyme. After you are through you
can screen for the loss of the marker you added.
In article <bpmurray*STUFFER*-0502990055310001 at macmac-2.ucsf.edu>,
>In article <79e41a$369$1 at justice.csc.cuhk.edu.hk>,
>s970854 at mailserv.cuhk.edu.hk (Andrew Leung) wrote:
>> Hi all,
>> Would there be possible to cut two restriction sites which are of 4
>> base pair apart (Not taking into account the restriction recognition
>It depends on the enzymes (although 4 bp is mighty close
>for even very tolerant enzymes). If you have access to
>an NEB catalogue then have a look in the back as it has
>a couple of tables on the efficiency of cutting when
>close to an end and lists a goodly number of their enzymes.
>A possible alternative is to linearise with one enzyme (A)
>and stick some kind of stuffer fragment in there. You
>then cut this construct first with the other enzyme (B)
>and after this is complete you cut with A. Removal of
>the stuffer fragment should mean success.
> Good luck,
>Bernard P. Murray, PhD
>Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA
More information about the Methods