How to use Expand PCR System

John Dixon jpcd0 at mole.bio.cam.ac.uk
Wed Sep 18 10:44:29 EST 1996


In article <Pine.A32.3.93.960918053951.80476A-100000 at kitts.u.arizona.edu>,
Stephen J Billington <sbilling at U.Arizona.EDU> wrote:

> On Tue, 17 Sep 1996, Andrew J. Doherty wrote:
> 
> > ouyhs at PUBLIC.CC.JL.CN wrote:
> > > 
> > > Dear Netter:
> > > 
> > >     I have bought a Expand Long Template PCR System and a Expand High
> > > Fidelity PCR System From Boehringer Mannheim. I wish I could use
> > > these kits to amplify and clone a gene about 10kb from bovine genome
> > > DNA. But I do not know which kind end be produced in the PCR products
> > > if we use those Expand systems.  Can I use the T-vctor to clone the
> > > PCR products? Do you have any literature on amplifing and cloning
> > > the DNA by using those Expand systems?
> > > 
> > >   Thank you for your help.
> > > 
> > >   My address is:
> > > 
> > >   Ouyang Hongsheng
> > >   Department of Biochemistry
> > >   Changchun University of Agriculture
> > >   175 Xian Road
> > >   Changchun 130062
> > >   Jilin, P. R. China
> > > 
> > >   Tel:86-431-7983451
> > >   Fax:86-431-7983442
> > >   Email: ouyhs at public.cc.jl.cn
> > > 
> > >   Yours Sincerely,
> > >   Ouyang Hongsheng
> > >   Ph.D.
> > > 
> > The data sheets for the Expand High Fidelity say that the enzyme
> > produces a mixture of blunt and T-overhang ends, like Taq. So youshould
> > be able to use T/A cloning. I haven't personally done T/A cloning with
> > this enzyme(s); we use it for it's proof reading in mutagenesis and cut
> > the ends of the PCR products.
> > 
> > Hope it helps.
> > 
> 
> Although you do get a mixture of blunt and A-overhang ends, the percentage
> of molecules with A-overhangs seems to be pretty small. I have had much
> more success with blunt cloning of these fragments than cloning into
> T-tailed vectors.
> 
> SJB 


Do you polish the ends as stratagene recommend for their PCRscript stuff
or do you use the expand stuff straight?

Cheers

John

-- 
John Dixon                                        Lab 44 (1223) 334131
Wellcome/CRC Institute                            Fax 44 (1223) 334134
Department of Genetics
Cambridge University    
United Kingdom                           e-m: jpcd0 at mole.bio.cam.ac.uk



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