Help???How to cip treatment with blunt-end vector???
dspinella at chugaibio.com
Fri Aug 15 12:46:12 EST 1997
> Does someone know how to CIP treatment with blunt-end vector???
> many thanks,
This is pretty basic stuff, Dong. You need to get your hands on a
recipe book like Maniatis or the Red Book if you're going to fool with
stuff like this, else you're just going to get in over your head real
fast. Anyway to answer your question, precipitate your vector with
ethanol, resuspend in Cip buffer (1mM ZnCl2, 1 mM MgCl2, 10 mM Tris-HCl,
pH 8.3) and add 1 U of CIP per pmole of blunt-ended linear vector.
Incubate reaction 30 minutes at 37 degrees (or just add Cip directly
your restriction digested DNA). Kill the enzyme by adding SDS to 0.5%
and EDTA to 5 mM and Proeinase K to 100 ug/ml. Incubate 30 minutes at
56 degrees. Cool the reaction to RT and phenol/chloroform extract and
then precipitate. BTW, you might look into shrimp alkaline phosphatase
rather than Calf Intestinal phosphatase as its easier to kill (by just
heat treatment). Its necessary to kill the enzyme prior to any ligation
else the residual A.P. will dephosphorylate your insert as well. Good
luck --D.G. Spinella
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