End conversion of PCR products

Hiranya Roychowdhury hroychow at NMSU.EDU
Fri Jan 16 15:41:24 EST 1998


At 02:15 PM 1/15/98 -0800, craig garen wrote:
>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
>
>

The polymerases and the kinases would work in PCR buffer, but the ligation
may not.
Now, for the home-made frugality: Yes, the price for the 25-rxn kit seems
high. This is what we (and most, I assume) routinely do-- 

1. Phosphorylate the primers to be used for PCR-- 
        The primers normally are resuspended in low TE buffer (10mM Tris
8/1mM EDTA). To an aliquot of this add kinase buffer to ~1x, 1.5x molar
excess of ATP and 1uL of kinase (~10U) (T4-PNK) and incubate at 37 C for
30-60 min. Kill the kinase by heating at 65 C/10 min. The resulting aliquot
of phosphorylated primers is ready for use as is w/o further purification. 

2. Run PCR reaction (alternatively, the phosphorylation may be carried out
directly after PCR reaction in the same buffer) 

3. Add 1uL more of 2.5mM dNTP per 50uL rxn, 1uL (~4U from BRL) of Klenow and
incubate for 15 min at 37 C; Heat-kill Klenow at 65 C for 10 min. This
produces "polished" amplicons. (Alternatively, use the dA overhangs of the
Taq generated products to clone in one of the TA vectors.)

4. Purify the blunt PCR product(s) from agarose gel and use in ligation.

Hope this is what you were interested in...


Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu




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