Dephosphorylating pACYM1

Alistair Forrest forrest at biosci.uq.oz.au
Thu Apr 7 21:31:10 EST 1994


G'day netters,
	Has anyone out there had trouble with the baculovirus
transfer vector religating despite dephosphorylation?
	My guess is the dephosphorylation isn't working properly for some 
reason ( we got back more transformants on our control than our ligation ).
	It's not the CIP (Calf intestinal Phosphatase) or carry over of
restriction enzyme and buffer as it worked fine for a pGEM ligation we were
doing at the same time.
	The only differences are that pACYM1 is a lot larger and that it
contains sequences for recombination into the baculovirus.

	The only things that I can think of that might be happening is that
the BamHI used for the digest ( or some other protein contaminant) is hanging
onto the ends and preventing access of the CIP.

	Any suggestions would be welcomed,
					Alistair Forrest




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