A scaled-down and simplified protocol for purifying recombinant Taq DNA polymerase

摘要

We previously described in a paper published in BIOS an undergraduate lab activity involving the gene cloning, expression, and purification of Thermus aquaticus (Taq) DNA polymerase, an enzyme used in the polymerase chain reaction (PCR). Based on the large number of requests for biological materials and questions about the protocols this paper invoked, we explored methods to simplify the protein purification portion of the published lab activity. A faster and simpler protocol would permit labs and classes with limited equipment or supplies the ability to produce active Taq DNA polymerase more easily for both teaching and research use. In the simplified protocol described here, bacterial cell lysis and enzyme purification is achieved in a small starting volume using only a hot water bath, a microcentrifuge, and one simple buffer. The purified enzyme from this protocol works well in PCR, and we additionally describe its use in a 2X master mix.