Microwave-assistant extraction method of recombinant Escherichia coli Pfu DNA (deoxyribonucleic acid) polymerase
A technology of recombinant Escherichia coli and extraction methods, applied in biochemical equipment and methods, enzymes, transferases, etc., can solve the problems of protein denaturation and inactivation, few, and loss of physiological activity, and achieve easy automatic control, good effect, Effect of reducing operation steps and time
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Embodiment 1
[0050] The Escherichia coli with recombinant Pfu DNA polymerase is induced and cultivated to obtain bacterial cells expressing Pfu DNA polymerase recombinantly, and then the following steps are adopted:
[0051] (1) Calculate the dry weight based on the determined water content (75%) of the wet bacteria, and add 0.03mol L according to the weight-to-volume ratio of the dry weight of the bacteria to the g / mL of the buffer solution as 1:10 -1 Phosphate buffer (pH5.0), then add 20 μg Tween20 at a mass volume percentage of 0.2% in g / mL and shake evenly until there are no bacterial clumps at the bottom.
[0052] (2) Add a magnetic rotor to the mixture, put it into a microwave reactor and start stirring at a speed of 500rpm.
[0053] (3) Select the microwave power of 700W, start microwave radiation, set the extraction temperature at 80-95°C, start timing when the temperature reaches 80°C, and continue to radiate, stop the radiation when the temperature reaches 95°C, and stop the radi...
Embodiment 2
[0056] The Escherichia coli with recombinant Pfu DNA polymerase is induced and cultivated to obtain bacterial cells expressing Pfu DNA polymerase recombinantly, and then the following steps are adopted:
[0057] (1) Calculate the dry weight based on the determined water content (80%) of the wet cells, and add 0.05mol L to the weight-to-volume ratio of the dry weight of the cells to the g / mL of the buffer solution as 1:40 -1 Tris-HCl buffer solution (pH 9.0), then add 480 μL SDS at a mass volume percentage of 1.2% in g / mL and shake evenly until there are no bacterial clumps at the bottom.
[0058] (2) Add a magnetic rotor to the mixture, put it into a microwave reactor and start stirring at a speed of 100rpm.
[0059] (3) Select the microwave power of 200W, start microwave radiation, set the extraction temperature at 70-90°C, start timing when the temperature reaches 70°C, and continue to radiate, stop the radiation when the temperature reaches 90°C, and stop the radiation whe...
Embodiment 3
[0062] The Escherichia coli with recombinant Pfu DNA polymerase is induced and cultivated to obtain bacterial cells expressing Pfu DNA polymerase recombinantly, and then the following steps are adopted:
[0063] (1) Calculate the dry weight based on the determined water content (78%) of the wet bacteria, and add 0.02mol L according to the weight-to-volume ratio of the dry weight of the bacteria to the g / mL of the buffer solution as 1:20 -1 Tris-HCl buffer solution (pH 8.0), then add 200 μL Triton X-100 at a mass volume percentage of 1.0% in g / mL and shake evenly until there are no bacterial clumps at the bottom.
[0064] (2) Add a magnetic rotor to the mixture, put it into a microwave reactor and start stirring at a speed of 300rpm.
[0065] (3) Select the microwave power of 500W, start microwave radiation, set the extraction temperature at 75-90°C, start timing when the temperature reaches 75°C, and continue to radiate, stop the radiation when the temperature reaches 90°C, an...
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