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TA cloning vectors (fwd)

berschic at convex.rz.uni-duesseldorf.de berschic at convex.rz.uni-duesseldorf.de
Tue Jul 5 08:09:29 EST 1994


In article <2v9aol$oqe at mserv1.dl.ac.uk> m-brazil at nimr.mrc.ac.uk writes:
> Forwarded message:
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>> Subject: TA cloning vectors
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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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