Help! Making this construct is driving me crazy!

[Klaus Salger]

Carlisle Landel (landel at helios) wrote:

: Hi!
: We are trying to ligate a 5kb Not I fragment into a Not I digested
: plasmid that is about 13 kb in size.  The fragment is supposed to
: go into a Not site in the middle of a 10 kb piece of mouse DNA that
: is cloned into Bluescript II (from which the Not site has been 
: removed).  
: This should be straightforward, but it isn't working.  Are we up 
: against some sort of size limit?
: We've done controls that tell us that the bugs are competent, the ligase
: works, the CIP works, the restriction enzymes are cutting.
: We just aren't getting any inserts.
: Does anybody have any suggestions?
: Thanks,
: Carlisle Landel

Size limitation should not be the problem. I did a similar construction
(6,4kb insert into 13,4kb vector, using XL1) without having trouble to
get it.
Did I get it right - another vector/insert combination treated with the
same enzymes and buffers worked? What about combinations of another vector
+ your 5kb insert and your 13kb vector + another insert?

Ciao
  Klaus

--
Klaus Salger                phone : ++49 (0)89 5902 -502
Zoologisches Institut       FAX   :                 -450
AG MacWilliams              e-mail: salger at zi.biologie.uni-muenchen.de
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