How to use Expand PCR System

Stephen J Billington sbilling at U.Arizona.EDU
Wed Sep 18 07:42:45 EST 1996


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 

------------------------------------------------------------
Stephen Billington Ph.D.
Department of Veterinary Science
University of Arizona
sbilling at U.Arizona.edu 
billing at vetsci.microvet.arizona.edu





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