PCR cloning - PAGE

at at
Thu Aug 24 18:59:42 EST 1995


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.



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