Help - cloning into dephosphorylated plasmid vector
rafael at corona.med.utah.edu
Fri May 26 15:48:14 EST 1995
On 25 May 1995, Hiranya Roychowdhury wrote:
> On 25 May 1995, Aron B. Jaffe wrote:
> > I agree with your friend - forget dephosphorylating the ends...
> > there's no need to do that with sticky end ligations...
> > aron
> That is not quite correct, what you said there, Aron. In fact,
> dephosphorylation should only be left out if the vector is cut with two
> enzymes generating non-compatible ends.
That's wrong. I normally make my ligations without dephosphorilation and
they work well. I got 30% of colonies with insert, and when you have some
kind of screening/selection, who cares about dephos? Or, in 10 minipreps
you can have three insert clones.
I only dephos when I need extremely efficient ligations (>90%), i.e.,
libraries, very long inserts, etc.
Rafael Maldonado | "y es que...
room 6160 Eccles Institute of Human Genetics | habemos gente pa to'"
Department of Human Genetics |
University of Utah |
Salt Lake City, Utah 84112. USA. | Joselito "El Gallo"
Rafael at genetics.med.utah.edu |
Rafael at corona.med.utah.edu |
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