Creating T-cloning vector

Rich Dudley rdudley+ at pitt.edu
Thu Feb 18 08:41:47 EST 1999


Dr Antony Halsall wrote:

> Hi
>
> I am trying to produce a T-cloning vector for the direct ligation of a
> PCR product into a reporter plasmid (pGL3-basic). I know it is possible
> to create the necessary 3' T overhangs by incorporating two adjacent
> restriction sites for either HphI, AspEI or XcmI, however all these
> enzymes will cut pGL3-basic. So my question is does anyone know of
> another enzyme that will allow the creation of 2 3' T overhangs or a
> method for cutting at a site present in the pGL3-basic and filling in to
> create the appropriate overhang?
>
> Thanks in advance
>
> Dr A. Halsall
> Dept. Psychological Medicine
> UWCM

There are many, may papers on this, as well as much discussion in this
newsgroup.  You might try a Medline search (www.ncbi.nlm.nih.gov), or go to
www.bio.net and search the archives if this newsgroup.

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.

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
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