Help???How to cip treatment with blunt-end vector???

C.Rodemer 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,
@chugaibio.com wrote:

> > 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.

I think many things in the Maniatis book are unnecessary,old and time
-consuming.  

>  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.

bye 
C.R.

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