if two estriction sites too close

[David Micklem]

If what you want to cut is in a circular vector, another alternative is to
cut two different aliquots with (enzyme A + enzymeC) and (enzyme B + enzyme
C) where C is a unique cutter in the vector, somewhere distant from A and
B.

For example, pBluescript and many other AmpR vectors have a unique AlwNI
site in the  ampicillin resistance cassette. You can cut with eg EcoRV and
AlwNI and purify the larger piece; and with HindIII and AlwNI and  purify
the smaller piece. Then do a 3-way ligation with your Blunt-HindIII insert.

Its much simpler (and works much better) than it sounds. An extra advantage
is that the fragments you want to purify are a very different size from the
single-cut vector in each case. So your background of re-ligated vector
goes way down.

HTH,

David


>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

-- 
D.R. Micklem,                            Time flies like an arrow... 
Beckman Institute,                   Fruit flies like a banana.       
Stanford, Ca 94305 USA           Email:dmicklem at cmgm.stanford.edu
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