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TA cloning vectors (fwd)

m-brazil at nimr.mrc.ac.uk m-brazil at nimr.mrc.ac.uk
Mon Jul 4 10:44:21 EST 1994


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> In article <199407022309.QAA24423 at net.bio.net> hwyckoff at ASRR.ARSUSDA.GOV ("HERBERT WYCKOFF") writes:
> >
> >In the not too distant past,  I seem to remember a posting of a method
> >for producing "TA" vectors by modifying blunt cut vectors with Taq
> >polymerase.  If someone would please send me a copy of the protocol,
> >I would appreciate it.
> >
> >Thanks
> >
> >Herb Wyckoff
> >HWYCKOFF at ASRR.ARSUSDA.GOV
> >
> >
> Hi Herb,
> I read something of tailing a blunt end vector with a ddTTp. 
> That adds only one nucleotide and this vector could be used 
> for cloning PCR products. So a normal tailing protocol with 
> ddTTP instead of dTTP should work.
> Peter
> 
> 
> 
Dear Herb and Peter and others who are interested,

I have a ref for constructing T-vectors (Marchuk et al, NAR, 19 no 5,
p1154). It uses dTTP alone with taq polymerase, which results in a single
thymidine at the 3' end of the vector. (If you use ddTTP you won't get
ligation to your PCR product unless you add phosphates to the end of the
latter.)  
 
You can use this principle to make blunt end ligations more efficient -
incubate insert with taq and dATP, vector with taq and dTTP.

I'm trying it right now, so i hope it works

Melanie
NIMR
Mill Hill, London, UK





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