Cloning PCR products?

kim at m44.unm.edu kim at m44.unm.edu
Thu Jul 23 12:09:36 EST 1992


In article <1992Jul23.030739.10401 at menudo.uh.edu>, bchs1b at Elroy.UH.EDU (Michael Benedik) writes:
>
>	There are a lot of different methods around for cloning PCR 
>products to get around the problems of the fragment ends left after 
>amplification. Does any one know whether this is solely a problem with
>TAQ polymerase? Are there other polymerases which leave nice ends
>suitable for subsequent cloning without going through the standard
>gyrations?


Nucleic Acids Research 19:184 (1991)
"Improved cloning efficiency of polymerase chain reaction (PCR) products after
proteinase K digestion."

Disclaimer:  I never tried this . . . I just collect them.
  This method is based on the hypothesis that the polymerase remains attached
to the end of the DNA, and is resistant to removal by standard methods.  They
do a proteinase K digestion after phenol and chloroform extraction and ethanol
precipitation.  then there was a second phenol chloroform extraction and
ethanol precipitation . . . what a lot of steps!  They show vastly improved
cloning efficiency.  I hope this is of some help.

Give us a follow-up if it works.

Daniel Kim
KIM at FLOVAX.LANL.GOV



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