Subcloning/CIP Question.

Michael Cooley szcooley at dale.ucdavis.edu
Mon Feb 6 11:53:40 EST 1995


Scott Stoughton (Scott_Stoughton at Sequana.com) wrote:
: Trying to shotgun subclone cosmids into pSPL3 splicing vector (exon trapping).
: Question: any advice on phosphotasing so the damn thing will ligate?
: Tx. s>
: Post here or email
: Scott_Stoughton at sequana.com

I have had good success following the method in the Boehringer Mannheim 
catalog. Essentially, you add 1/10 vol 0.2M EGTA after the CIP reaction 
to destablize the enzyme, then you do a phenol/chloroform extraction and 
precip. The CIP is very tenacious and will prevent ligation is not removed.


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