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MOLECULAR BIOLOGY: WORKING WITH DNA

ENZYMATIC MANIPULATION

Preparation and Purification of Taq Polymerase

Preparation and Purification of Taq Polymerase Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Contributor:	Simon Dawson at the School of Biomedical Sciences, Queen's Medical Centre
Overview Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
You will need a bacterial cell line that expresses the Taq polymerase and is ampicillin resistant. The procedure takes four days from start to finish and generally results in approximately 15,000 Units of Taq.
Procedure Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
A. Day One 1. Inoculate overnight two 2-liter flasks containing 500 ml of TB/Amp Solution with 15 ml of Taq Polymerase producing bacteria (see Hint #2). 2. Grow the culture to an Optical Density at 600 (OD600) of 0.6 (see Hint #3). 3. Add Isopropyl β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM (0.119 grams IPTG per liter of TB/Amp Solution). 4. Grow the culture overnight (do not exceed 16 hr). B. Day Two (unless otherwise noted the remaining steps should be carried out on ice or at 4°C) 1. Pellet the 500 ml of cells by centrifugation at 3,500 X g for 15 min and discard the supernatant. 2. Resuspend the pellet in 40 ml of Buffer A. 3. Add an equal volume of Buffer B and incubate at 75°C for 1 hr (mix the tube periodically). 4. Centrifuge at 8,000 X g for 15 min at 4°C. 5. Save the supernatant and add 1.86 mg KCl per ml of supernatant. 6. Remove 150 ml of DP-1 Cation Exchange Resin (packed volume) to a conical centrifuge tube. 7. Add an equal volume of sterile ddH2O, mix well, and centrifuge at approximately 3,000 X g to pellet the resin. 8. Discard the supernatant. 9. Repeat the washing of the resin on more time with ddH2O (Step #B7 to #B8). 10. Add an equal volume of ice-cold Buffer B to the resin, mix well, and centrifuge at approximately 3,000 X g to pellet the resin. 11. Discard the supernatant. 12. Repeat the washing of the resin with ice-cold Buffer B three more times (Steps #B10 to #B11). 13. Aliquot the washed resin equally (75 ml each) into two 250 ml conical centrifuge tubes. 14. Aliquot equal volumes of the supernatant (prepared in Step #B5) to each of the conical centrifuge tubes containing resin (prepared in Step #B13). 15. Vortex the tubes well. 16. Incubate on a shaking platform for 30 min at 4°C. 17. Centrifuge to pellet the resin at approximately 3,000 X g for 2 min at 4°C. 18. Discard the supernatant. 19. Add 100 to 200 ml of ice-cold Buffer B and mix well. 20. Centrifuge to pellet the resin at approximately 3,000 X g for 2 min at 4°C. 21. Remove the supernatant by aspiration and discard the supernatant. 22. Repeat the washing of the resin three more times (Steps #B19 to #B21). 23. Add one packed-bed volume of ice-cold Buffer C to the washed resin and mix well. 24. Centrifuge to pellet the resin at approximately 3,000 X g for 2 min at 4°C. 25. Carefully remove and save the supernatant. 26. Repeat the elution of protein from the resin two more times (Steps #B23 to #B25). 27. Add 15 g of Ammonium Sulfate ((NH4)2SO4) per 50 ml of collected supernatant while stirring rapidly (see Hint #4). 28. Centrifuge at 15,000 to 17,000 X g for 10 min at 4°C. 29. Discard the supernatant and resuspend the pellet (which is thinly translucent) in 25 to 35 ml of Buffer C. 30. Dialyze the resuspended pellet against 2 liters of Dialysis Buffer at 4°C for between 6 to 18 hr. Repeat at least once. C. Day Three 1. Determine the polymerase activity by preparing serial dilutions of the isolated Taq and compare that to a commercially prepared Taq. 2. Aliquot concentrated Taq polymerase at desired concentration in Dilution Buffer and store the aliquots at -20°C.
Solutions Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Buffer B    0.5 mM PMSF* (CAUTION! See Hint #1) 0.5% (v/v) Tween-20 *Add just before use 1 mM EDTA 50 mM KCl 20 mM HEPES, pH 7.9 0.5% (v/v) NP-40 Buffer A    1 mM EDTA 50 mM Dextrose 50 mM Tris, pH 7.9 TB/Amp Solution    12 g Tryptone 4 ml Glycerol Add appropriate concentration of Ampicillin for Taq polymerase expression Add ddH2 to 900 ml Add 100 ml sterile 0.72 M Potassium Phosphate, Dibasic (K2HPO4) Add 100 ml sterile 0.17 M Potassium Phosphate, Monobasic (KH2PO4) 24 g Yeast Extract Dilution Buffer    100 mM KCl 50% (v/v) Glycerol 20 mM HEPES, pH 7.9 0.1 mM EDTA Dialysis Buffer    100 mM KCl 0.5 mM PMSF* (CAUTION! See Hint #1) 50% (v/v) Glycerol *Add just before use 1 mM DTT* 1 mM EDTA 20 mM HEPES, pH 7.9 Buffer C    0.5 mM PMSF* (CAUTION! See Hint #1) 200 mM KCl 0.5% (v/v) Tween-20 *Add just before use 1 mM EDTA 20 mM HEPES, pH 7.9 0.5% (v/v) NP-40
BioReagents and Chemicals Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Isopropyl beta-D-thiogalactopyranoside Potassium Phosphate, Monobasic Yeast Extract Tryptone Dextrose Ampicillin NP-40 DTT EDTA Ammonium Sulfate Glycerol Tween-20 PMSF Tris HEPES Potassium Phosphate, Dibasic Potassium Chloride
Protocol Hints Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions. 2. The volumes can be scaled down. 3. Culture should be collected at approximately the mid-log phase. 4. It is advantageous to aliquot the supernatant collected from the resin into 50 ml conical or round-bottom centrifuge tubes.
Citation and/or Web Resources Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
1. Engelke DR, Krikos A, Bruck ME, Ginsburg D. Purification of Thermus aquaticus DNA polymerase expressed in Escherichia coli. Anal Biochem. 1990 Dec;191(2):396-400. 2. Pluthero FG. Rapid purification of high-activity Taq DNA polymerase. Nucleic Acids Res. 1993 Oct 11;21(20):4850-4851.

   

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