TA Vector

Martin Leach leach at mbcrrc.bu.edu
Fri Oct 28 14:19:17 EST 1994


In article <1994Oct28.154644.1 at molbiol.ox.ac.uk>, jnewton at molbiol.ox.ac.uk
wrote:

> Hi,
> 
> I'm looking for a method of preparing a homemade TA type vector. I seem to 
> recall somebody posting here last year sometime that this was possible by 
> using a blunt cutter, and adding the T overhangs with either Taq or Terminal 
> Transferase. If anybody has either protocol, or can remember when said
article 
> was posted (so I can find it in the archive), I'd be eternally greatful
if the 
> would mail me!
> 
> Thanks,
> 
> Justin                                                  
> jnewton at molbiol.ox.ac.uk
> -- 
>    ***************************************************************************
>    *                                                                         *
>    *   I'm suffering from Mood Poisoning.... It must be something I hate!    *
>    *                                                                         *
>    ***************************************************************************

Digest vector e.g. pBluescript with blunt cutters (Eco RV) run out on gel
and cut out linearised vector. Precipitate DNA then incubate with Taq pol
for 2 hours (in standard PCR buffer with dTTP alone). Precipitate....
resus,and use..
we start with 5 ug of vector (and resus in 50 ul at very end) use 5-10 ul
in ligations.

Martin

-- 

.....          Martin Leach                Email:leach at mbcrrc.bu.edu 
   _|_______      Dept. of Pharmacology       Phone: (617) 638-5323        
   /  o    /      Boston Univ. School of Med. Fax:   (617) 638-4329         
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