Creating T-cloning vector

Chris Kafer ckafer at iastate.edu
Thu Feb 18 13:37:29 EST 1999


On Thu, 18 Feb 1999 08:41:47 -0500, Rich Dudley <rdudley+ at pitt.edu>
wrote:


>Basically, you can cut with any blunt ender (SmaI, EcoRV, etc.), and
>incubate with Taq and dTTP (only!) for 1-2 hrs at 72oC.  Having tried this
>myself, and judging from comments in the newsgroups, if you're doing
>something important, it might be better to invest in a kit, since they are
>much better than the homemade stuff.
>

I think there was a paper (Biotechniques??) that compared the
efficiency of T addition using differing blunt cutters.  If memory
serves SmaI was not as "good" as EcoRV.

Havent done the controls but I never have a problem with the homemade
T vector.  It's extremely simple to make and much cheaper.  You can
cut down on background by religating your tailed vector and then gel
purifying any religated away from unreligated (also Biotechniques??)

-Chris


>rich
>
>--- --- --- -- -- -- --- --- ---
>Richard J. Dudley (rdudley+ at pitt.edu)
>Research Specialist V
>Dept. of Cell Biology and Physiology
>University of Pittsburgh
>http://www.cbp.pitt.edu
>---> search BIONET archives at http://www.bio.net <---
>
>




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