if two estriction sites too close

[John R. McQuiston]

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.  

John



In article <bpmurray*STUFFER*-0502990055310001 at macmac-2.ucsf.edu>, 
bpmurray*STUFFER*@socrates.ucsf.edu says...
>
>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 
>> sequence)?
>> Andrew
>
>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
>-- 
>Bernard P. Murray, PhD
>Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA