PCR cloning - PAGE

[Francisco G da Nobrega ib - bio 7588]

On 22 Aug 1995, 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?
> 
> 
We learned recently in this section that proteinase K treatment of PCR 
products can increase very much the efficiency of the subsequent 
enzymatic treatments making the DNA clonable. Reference is Crowe etal. 
Nucleic Acids ResearchL 19:184 We are trying this new treatment. Good 
luck to all of us...
				F.G.Nobrega  Biologia/IBUSP
				fgdnobre at usp.br