TA cloning vectors

berschic at convex.rz.uni-duesseldorf.de berschic at convex.rz.uni-duesseldorf.de
Tue Jul 5 08:32:16 EST 1994


>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
>
>
Hi Melanie,
I found the citation for the ddTTP vectors for cloning. It is :
Holton et al., 1991: A simple method for direct cloning of PCR-products 
using ddT-tailed vectors. NAR 19:1156.

I didn´t use it but why shouldn´t it work? The ligation of 
dephosphorylated vector (to inhibit self ligation) with PCR product does
also work. The 5´end of vector could ligate with 3´ end of PCR-product. 
Perhaps the not ligated 3' end of vector (ddT) could be repaired by 
excision and replacing of ddT during replication in the host. 

Peter
Uni Duesseldorf
Duesseldorf, Germany



More information about the Methods mailing list