Simple and efficient method for preparing and purifying TaqDNA polymerase

Abstract

The invention provides a simple and efficient method for preparing and purifying TaqDNA polymerase. The method includes: 1) establishing a TaqDNA polymerase expression vector pET-30aTAQ; 2)converting the TaqDNA polymerase expression vector pET-30aTAQ on a kanamycin low salt LB plate, obtaining a bacterial strain BL21 (DE3) / pEP-30aTAQ; 3) inoculating in a kanamycin low salt LB fluid nutrient medium, and performing shake cultivation until OD600=0.4-0.9; 4) performing isopropyl-beta-d-thiogalactoside (IPTG) induction for 2-24 hours; 5) collecting supernatant of the medium in centrifugal mode; 6) heating the supernatant for 10 minutes at the temperature of 80-100 DEG C, and performing ice-bath for 1 minute at the temperature from 0 DEG C to -20 DEG C; 7) collecting supernatant in centrifugal mode; and 8) freezing enzyme liquid after detection so as to obtain finished enzyme. The simple and efficient method has the advantage that purification is finished through two-step centrifugation and one-step thermal circulation, and the method is simple. The simple and efficient method for preparing and purifying the TaqDNA polymerase omits a bacteria cracking process and subsequent multifarious purification steps of a traditional method, improves purification of the TaqDNA polymerase, reduces pollution, saves preparation cost, shortens production cycle, and can be applied to continuous online immobilized production of the TaqDNA polymerase.

Description

technical field [0001] The present invention relates to the field of molecular biology, a Taq DNA polymerase (Thermus aquaticus DNA polymerase) expression and purification technology, especially a simple, efficient and low-cost method for purifying Taq DNA polymerase, which is especially suitable for a large number of low-cost commercial Production and preparation of Taq DNA polymerase. Background technique [0002] PCR, also known as polymerase chain reaction, is an in vitro nucleic acid amplification technique developed in the mid-1980s. During thermal cycling, it uses high temperature resistant polymerases and complex substrates to rapidly amplify DNA fragments in a short period of time. It has outstanding advantages such as specificity, sensitivity, high yield, rapidity, simplicity, good repeatability, and easy automation. These advantages make PCR technology one of the most important tools in molecular biology, and is regarded by many scientists as the most important ...

AI Technical Summary

Problems solved by technology

These methods are cumbersome and time-consuming, and the chromatographic media and gels used in the process of removing impurities are all expensive, resulting in high costs. The purification process reduces the yield and enzyme activity of polymerase finished enzymes, so the preparation and The process of purifying Taq DNA polymerase needs to be improved urgently

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