Looking for a pBluescript with KAN selection or...

[John Dixon]

In article <62uihd$9tp$1 at nntp3.u.washington.edu>,
polliwog at u.washington.edu (S. Findley) wrote:

> Has anyone out there constructed a pBluescript with Kanamycin resistance
> instead of Ampicillin?
> 
> There is a pesky Eam1105I/AhdI restriction site at position ~2046 in the
> Amp resistance gene in pBS that I either need to mutate in the Amp or
> replace the cassette with th Kanamycin resistance gene.
> 
> Any tips appreciated.
> 
> (Does anyone out there know how companies selling T-vectors are making the
> 3' T-overhangs efficiently? I was planning on making the T-vector with 2
> AhdI/Eam1105I resitriction sites in the polylinker til I noticed the site
> in the Amp resistance gene. Perhaps a company has already mutated that
> site?)
> 
> 
> -Thanks
> -- 
> Seth D. Findley               polliwog at u.washington.edu
> Department of Biochemistry    Phone: 206-543-1710
> Health Sciences Building J579
> University of Washington, Box 357350, Seattle, WA 98195



I made one with 2 XcmI sites (not present in pBuescript) but it didn't
work very well. NEBs info indicates that XcmI should be purer than AhdI ie
it's cloned + better cutting/religation data but it still didn't work for
me - loads of blues, whites incorrect nibbled vector.

You could use pCRscript camR - there's no AhdI in that.

Good luck,

JD

-- 
John Dixon                                        Lab 44 (1223) 334131
Wellcome/CRC Institute                            Fax 44 (1223) 334134
Department of Genetics
Cambridge University    
United Kingdom                           e-m: jpcd100 at mole.bio.cam.ac.uk