Help! Making this construct is driving me crazy!

[Martin Leach]

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

1. try dephosphorylating insert or vector...not both
2. use two enzymes.....Not I and something else to prevent
concatamerization
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 digest..to enrich)

Hope you understand this...

Martin



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