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

( Andy.Law @ bbsrc.ac.uk )
( Big Nose in Edinburgh )



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