Sma I religation problem

[Mad Dan Eccles]

In article <3ujkm8$6h1 at hydra.acs.ttu.edu>, Amadou Ba <PHYASB> writes:
> 
> We're doing EcorI and SmaI to insert a fragment digested with
> EcorI and StuI (blunt end).... Each enzyme linearize the corresponding
> vector when used alone. But like in the original article we get to 
> many colonies from the control ligation (no insert).

Therefore either one of the two (or both:() of the digests is significantly 
less than 100% effective.  I don't know exactly what that means, but if you 
assume competence at 10E7 cells/ug DNA, you can work it out for your expt.

You will get this problem if the second enzyme is too close to the end of the 
linearized vector.  Check the NEB catalogue to see if this is a problem for 
your enzymes.

> If we understand correctly, adding Klenow and dNTP will make 
> every thing blunt ended.
> 

Should still work, but you need to phosphatase the vector.


Richard
-- 
Richard P. Grant MA DPhil      rpgrant at molbiol.ox.ac.uk
Nuffield Department of Obstetrics and Gynaecology, University of Oxford.
FAX +44 1 865 69141            TEL +44 1 865 221018
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"It was only a _small_ thermonuclear device."