A simplified protocol for producing Taq DNA polymerase in biology laboratory

Research in Molecular Medicine

Touraj Farazmandfar¹  Alireza Rafiei²  Reza Valadan²  Mohammad Bagher Hashemi-Sotehoh²  Mohammad Alavi³  Fatemeh Moradian³ 

[1] Faculty of Advanced Medical Science Technologies, Golestan University of Medical Sciences, Gorgan, Iran;Molecular and Cell Biology Research Center, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran;Sari Agricultural sciences & Natural Resources University, Sari, Iran;

关键词: Taq polymerase;    expression;    Purification;   

DOI  :  

来源: DOAJ

【 摘 要 】

Background: Taq DNA polymerase is a very important enzyme for molecular biological studies such as DNA amplification and DNA sequencing by the PCR. It is a standard enzyme that is used in 90% of molecular biology labs today. The aim of this study was to produce Taq DNA polymerase enzyme in E. coli by a reliable, practical, simple and low cost method.Materials and Methods: Inthisstudy,theTaqgenewasamplifiedfromthegenomicDNAofThermus aquaticus and clonedinto pTrc99A vector. Recombinant plasmid is expressed in E. coli strain TOP10. Product protein is extracted and purified. Expression of gene was analyzed by SDS-PAGE and gene amplification.Results: SDS-PAGE showed an approximately 94 KDa protein. The density of protein bands in agarose gel electrophoresis indicated that the purified enzyme is more active than the non purified one.Conclusion: The protocols described in this paper lead to the production of pure and active enzyme that can be applied in both teaching and research laboratories.

【 授权许可】

Unknown