CIPPers for breakfast

Anthony Tomlinson anthony at pplros.demon.co.uk
Fri Jul 22 09:30:58 EST 1994


Hi:

I am trying to clone a ClaI fragment in to a ClaI site of a
pBR322-derived vector, and am treating the vector with NEB's CIP at 10U
of CIP to 5yg of 10Kb fragment, in 20yl of 1x Boehringer H buffer, for 2
h at 37!C, followed by gel purification (Prepagene). It's not working. 
Work with control fragments and vectors suggests that the ends are
damaged, either in the ClaI digest (doubtful) or in the CIP step.  Does
anyone know if CIP (especially NEB's) damages 5' overhangs?  I have
previously CIPped blunt ends with no trouble.

Thanks,
Anthony Tomlinson
anthony at pplros.demon.co.uk



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