Background:
Thermostable DNA polymerase (Taq Pol Ι) from
Thermus aquaticus
has beenwidely used in PCR,
which was usually extracted with Pluthero's method. Themethod used ammonium sulfate to precipitate
the enzyme, and it saved effort and money but not time. Moreover, we found that 30–40% activity
of Taq Pol I was lost at the ammonium sulfate precipitation step, and the product contained a small
amount of DNA.
Results:
We provided a novel, simplified and low-costmethod to purify the Taq Pol Ι after overproduction of
the enzyme in
Escherichia coli
, which used ethanol instead of ammonium sulfate to precipitate the enzyme.
The precipitate can be directly dissolved in the storage buffer without dialysis. In addition, DNA and RNA
contamination was removed with DNase I and RNase A before precipitation, and the extraction procedure
was optimized. Our improvements increase recovery rate and specific activity of the enzyme, and save
labor, time, and cost.
Conclusions:
Our method uses ethanol, DNase I, and RNase A to purify the Taq Pol Ι, and simplifies the
operation, and increases the enzyme recovery rate and quality.
