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
> 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.
More information about the Methods