PCR cloning - PAGE

J. Morrey jmorrey at cc.usu.edu
Tue Aug 22 22:56:24 EST 1995


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?



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