PCR cloning - PAGE
Andy Law Big Nose
Andy.Law at bbsrc.ac.uk
Fri Aug 25 08:34:49 EST 1995
In article <-2408951700380001 at mac67123.salk.edu>, @ wrote:
> In article <1995Aug22.215624.59506 at cc.usu.edu>, jmorrey at cc.usu.edu (J.
> Morrey) wrote:
> > We have tried unsuccessfully to clone 3.0 and 4.4 kb PCR products into
> > plasmids. We have employeed many different strategies including
> > blunt-end ligation after Klenow fill-in and kinase reactions,
> > fractionation of the PCR product by agarose followed by restriction
> > cutting and ligation into the vector, blunt-end ligation after agarose
> > fractionation, etc. We have empirically observed that agarose-purified
> > PCR product (via agarase enzyme) may be inhibited by perhaps
> > contaminants, thereby yielding no cloned DNA. Consequently, we are
> > considering fractionation by PAGE and subsequenct purification by
> > elution. Here are the questions...
> > 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?
> Two suggestions;
> 1. Try TA-cloning. It's worked great in my hands. There are several
> comercial kits available for this, or you can make your own vector.
> 2. Use something like FMC's GTG agaroses (genetic technology grade). Any
> of their products with a GTG suffix is certified for use in cloning
> experiments (i.e. will not inhibit various enzymes). I do all in-gel
> cloning using seaplaque gtg or nusieve gtg and never have any problems,
> nor do I ever worry about recovering things from gels.
Third suggestion. Blast everything with Proteinase-K and then PCI extract
prior to cloning. Improves efficiency no end.
( Andy.Law @ bbsrc.ac.uk )
( Big Nose in Edinburgh )
More information about the Methods