Help - cloning into dephosphorylated plasmid vector

[Rafael Maldonado]

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
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Rafael Maldonado                             |  "y es que...	  
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