Help - cloning into dephosphorylated plasmid vector
hroychow at NMSU.EDU
Mon May 29 10:35:16 EST 1995
On Fri, 26 May 1995, Rafael Maldonado wrote:
> 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 |
> Tel: 801-581-4429 |
> Fax: 801-585-3910 |
Yes, but I always aim for 'very efficient ligations' and with
dephosphorylated vectors, I routinely obtained over 90% positive clones
in my ligations. For a cDNA library, that kind of difference in
efficiency could make or break a graduate student's research.
Hiranya S. Roychowdhury
Plant Genetic Engineering Lab.
Box 3GL, NM State Univ.
Las Cruces, NM 88003
Phone: (505) 646-5785
hroychow at nmsu.edu
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