Help! Making this construct is driving me crazy!

Martin Leach leach at
Tue Jan 10 11:31:00 EST 1995

In article <3es6fl$8o5 at>, landel at helios (Carlisle Landel)

> 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

1. try dephosphorylating insert or vector...not both
2. use two enzymes.....Not I and something else to prevent
3. design experiment so that ligation abolishes site in vector...then
redigest ligation reaction to digest the concatamers...and repeat
ligation....(e.g. clone not I fragment into...another enzyme site so that
ligation abolishes the other enzyme site...then enrich)

Hope you understand this...



.....          Martin Leach                Email:leach at 
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