if two estriction sites too close
dmicklem at cmgm.nospam.edu
Fri Feb 5 14:58:10 EST 1999
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
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.
>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
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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