Which should be CIPed, 7K one or 3K one?

Richard P. Grant see_sig at cmtech.co.delete.uk
Wed Aug 26 02:09:35 EST 1998


In article <199808260340.LAA11180 at mail.shcnc.ac.cn>, shengyang at USA.NET wrote:

: 
: What you thought is not the point of the question. Because, the two
: fragments are both cut out by ~ScaI~. You know, ScaI cut ampR into 2 parts.
: So, the 3k one carries one part of ampR and the 7k one carries another part
: of it. Then which one is the vector and which one is the insert? I think it
: is hard to say.
: 

Hah.  Sneaky.

: One of the fragments contains the ori, but neither contains the ampR. If
: re-circularized and up-taked by the competent cells, one can not replicate
: and the other can but its host can not grow on LB-Amp plate. So, I consider
: that the background is rather low. The transformants which appear on the
: plate should contain recombinant plasmid very likely.
: 

Good one.  I'd suggest de-P -ing the frag containing ori. You'll get some
losses but I guess if you titrate so that the non-dephosphorylated frag is
in excess, you'll increase your chances.

 And I'd use USB's Shrimp alk phosph in preference to CIP; it seems to be
as efficient and is completely deactivated at 70C.

HTH

R

-- 
Richard P. Grant MA DPhil     |             rgrant at cmtech.co.uk
Senior R&D Scientist          |             work: www.cmtech.co.uk
Cambridge Molecular           |      home: www.avnet.co.uk/adastra           
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