Help - cloning into dephosphorylated plasmid vector

Hiranya Roychowdhury 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.
> 
> 
> 
> Rafa
> ___________________________________________________________________
>                                              |
> 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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