End conversion of PCR products

craig garen craig_garen at HOTMAIL.COM
Thu Jan 15 17:15:40 EST 1998


Hi all

    Novagen sells a kit called the Perfectly Blunt PCR Blunt Cloning
System which includes a so-called "PK" enzyme mix for  end polishing and
phosphorylation.  Can anyone suggest a ratio of the two enzymes (ie T4
DNA polymerase and T4 PNK) to use.  Also, apparently a 10X buffer is
included, any ideas on the composition of this for it to be compatible
for both enzymes and for T4 DNA ligase (T4 lig is added directly to the
end conversion with the dephophorylated vector following heating to kill
T4 PNK, thus I assume all three must be compatible in the same buffer)?

    Is anybody sick and tired yet of cutsie-named products which, if
they even have a hint of molecular biology application, we end up paying
ridiculous amounts for?  This kit (25rxns) is being sold for >$250US.
Something tells me I can do the home-made thing without the dumb handle
for a whole lot less.

Thanks in advance for your advice

Craig Garen




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