On Fri, 22 Jul 1994 14:30:58 GMT, Anthony Tomlinson wrote:
>>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.
>anthony at pplros.demon.co.uk
Use less CIP. You need as little as 0.01U / pmol DNA ends. Also, you should
run the reaction in an appropriate buffer. To my knowledge CIP requires
Zn++ Ions, which are not contained in the Boehringer H buffer.