Protocol Taq Polymerase Preparation

4.8. Medium

4.8.1. 2 x YT medium Per liter: Tryptone 16g Yeast extract 10g NaCl 5g Adjust pH to 7.0 with 5N NaOH. Sterilize by autoclaving.

4.8.2 LB medium Per liter: Tryptone 10g Yeast extract 5g NaCl 10g Adjust pH to 7.0 with 5N NaOH. Sterilize by autoclaving.

4.9. Buffers:

4.9.1. Buffer A: 50 mM Tris-HCl, 50mM dextrose and 1 mM EDTA, pH 7.9

5. Procedure

5.1. Grow and collect the Cell:

5.1.1. Plate the E.coli strain containing the Taq construct on LB agar containing 100ug/mL ampicilin. Incubate the culture overnight at 37oC

5.1.2. Pick up one fat colony from overnight plate, inoculate into 2 mL LB containing 100 ug/mL ampciling at morning, then transfer to 200 mL LB containing ampcilin at evening, and grow overnight.

5.1.3. Transfer 15 mL overnight culture to 1L 2xYT containing 100 ug /mL ampcilin at morning (6L every time), and grow at 37oC with good aeration to OD595 0.6-0.8, then add IPTG to a final concentration of 1 mL and grow the culture overnight.

5.1.4. Harvest cells by centrifugation at 3700 rpm for 30 min art 4oC using 500 mL centrifuge buck.

5.1.5. Resuspend the cell in Buffer A, and transfer to 50 mL conical tube, and collect the cell by centrifuge.

5.1.6. Weigh the cell pellet and record it. (70-80g is needed for purification)