Help???How to cip treatment with blunt-end vector???
c.rodemer at dkfz-heidelberg.nospam.de
Thu Aug 21 06:00:57 EST 1997
In article <33F4A548.5D90 at chugaibio.com>, dspinella at chugaibio.com,
> > 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
I think many things in the Maniatis book are unnecessary,old and time
> Anyway to answer your question, precipitate your vector with
> ethanol, resuspend in Cip buffer (1mM ZnCl2, 1 mM MgCl2, 10 mM Tris-HCl,
I cut my PBluescript with EcoRI and didnt precipitate anything.I just
added buffer for CIP and some Units CIP .Then incubate at 37C.
> 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.
Well, I just made two phenol/chlorof/IAA extractions and precipitated the
vector with 2 vol ethanol. Then I used it for ligation.
Obviously it worked. I screened only 10 cols and I found the recombinant
pBS in one colony.
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