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A simple and efficient method for extraction of Taq DNA polymerase

Author:
Chen, Sique, Zheng, Xiujuan, Cao, Hongrui, Jiang, Linghui, Liu, Fangqian, Sun, Xinli
Source:
Electronic Journal of Biotechnology 2015 v.18 pp. 355-358
ISSN:
0717-3458
Subject:
DNA, DNA-directed DNA polymerase, Escherichia coli, RNA, Thermus aquaticus, ammonium sulfate, biotechnology, deoxyribonuclease I, dialysis, enzyme activity, ethanol, labor, polymerase chain reaction, ribonucleases, thermal stability
Abstract:
Thermostable DNA polymerase (Taq Pol Ι) from Thermus aquaticus has been widely used in PCR, which was usually extracted with Pluthero's method. The method 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.We provided a novel, simplified and low-cost method 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.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.
Agid:
5526753