Making a TA Cloning Vector

Helge Weissig helgew at ljcrf.edu
Fri Jan 28 18:33:20 EST 1994


Rasmussen at bioscience.biology.utah.edu (Randy P. Rasmussen) wrote:

>I have heard that you can add a single 3' overhanging T to a blunt end by
>incubating your blunted DNA with Taq polymerase and dTTP.  This would be a
>dandy way to make your own PCR cloning vectors.  Can anyone confirm or deny
>this?  Does anyone have a reference?

the reference is: Marchuk et al., NAR 19:1154 (1991)

it worked beautifully, when we first used it, but when we ran out of
T-vector, we never got as good results (background, cloning efficiency) as
before. of course not! :)
for those of you without library:

        * digest vector w/ blunt-cutter (eg. EcoRV, SmaI)
        * incubate w/ Taq Pol (1U/ug of plasmid/20 ul vol) in the presence
          of 2 mM dTTP for 2 hrs @ 70oC (use the supplied buffer or 50 mM
          KCl, 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl2, 200 ug/ml BSA)
        * purify plasmid w/ your favorit method (Phenol/CHCl3 & EtOH ppt
          is enough)
        * ligate to PCR fragment o/n @ 14oC

cheers,
helge


Disclaimer: No, I don't work against Invitrogen.


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Helge Weissig      

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