PCR cloning - PAGE

John K. Troyer jtroyer at umabnet.ab.umd.edu
Wed Aug 23 08:45:33 EST 1995


On Wed, 23 Aug 1995, Julian Parkhill wrote:

> In article <1995Aug22.215624.59506 at cc.usu.edu>, jmorrey at cc.usu.edu (J.
> Morrey) wrote:
> 
> > Will polyacrylamide gel electrophoresis likely yield less inhibitory
> > contaminants which would lead to cloned PCR products?  Will 3.0 or 4.4
> > Kb fragments elute from the polyacrylamide during the purification
> > process?
> 
> It has certainly been my (subjective) experience that DNA purified from
> acrylamide is easier to clone than DNA from agarose, though I have never
> performed a controlled experiment.

Absolutely!   Products in the agarose can inhibit ligase activity as 
well. PAGE purification is definitely more pure, and better to use for 
purification

> 
> As for the purification itself - 3-4 kb is probably too big to elute by
> passive diffusion, but I'm sure you could get it out by electroelution.
> 


3-4 kb will electroelute out of a PAGE gel with no problem.  Just 
stain the gel well with EtBr prior to excision of the band and then 
monitor the electroelution periodocally by transillumination.

John



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