Wild-type Taq Purification

1. Transform BL21 (DE3) pLysS with FastTaq (pET30b-sso7d-Taq_d289, 377) plasmid.

2. Grow on LB/Kan/Chl plate o/n.

3.  Pick a colony, grow in 200 ml LB/Kan/Chl.

Optional: screen colonies for high yield expression.

4. Add 30 ml bacteria to 1 L Autoinduce Medium, Grow total of 6 L in 6 flasks at 30 ^oC or 37 ^oC O/N.

5. In the morning, spin down cells, wash cells with Buffer A or DI-water, spin. Weigh cell pellet, record weight.

6. Continue to grow cells for a few more days until total cell pellet is 100 g.

7. Add 25 ml Buffer B  to cell pellet from 1 L culture, suspend cells by votexing.

8. Freeze cell pellet with dry ice.

9. Thaw cell pellet at 75 ^oC (water bath).

10. Repeat freeze-thaw for 1 or 2 more cycles.

11. Sonicate lysate if it is still sticky.

12. "Boil" lysate at 75 ^oC for 30 min, cool on ice for 15 min.

13. Spin at 12000 rpm for 10 min at 4 ^oC (Sorvall high speed centrifuge), collect supernatant.

14. Transfer supernatant to ultracentrifuge tubes. Balance to 0.1 g.

15. Spin at 25000 rpm for 10 min at 4 ^oC, collect clarified supernatant into a clean 500 ml centrifuge tube.

16. Add ~30 g ammonium sulfate (NH₄)₂SO₄ slowly into the 100 ml of supernatant while stirring or mixing at room temperature.

17. Incubate at room temperature for 15 min.

18. Spin at 12000 rpm for 15 min at 4 ^oC.

19. Resuspend pellet in 5 ml Buffer A, wash centrifuge tube with up to 2 to 4 more times with Buffer A, combine washes. Try not to form bubbles.

20. Add saturated sterile (autoclaved) dibasic potassium phosphate, K₂HPO₄, (pH 10.3) to  Taq suspension to final concentration of 10%, i.e., add 1 ml to 9 ml of suspension. Mix well for several seconds. Then add an equal volume of ethanol to the 10% salt solution and mix vigorously for several seconds. Spin for 2 min. Collect the top (organic ethanol) phase containing the Taq.

Check the bottom (aqueous) phase for both Taq and nucleic acids by PCR and agarose gel.

At this step, the Taq in aqueous phase should be at least  1×10⁶ units from 1 L culture. (i.e., 1 unit/5 ng protein. -->1 million units/5 mg protein).

Optional: Before aquous two phase extraction, desalt Taq suspension with Buffer B by gel filtration (FPLC), Combined desalted fractions containing active Taq DNA polymerase.

21. Dilute Taq with Buffer C to 20% ethanol (1 ml Taq in ethanol + 4 ml Buffer C). 

21. Load Taq onto a 150-ml BioRex 70 (Bio-Rad) ion exchanger column, pre-equilibrated with Buffer C. Collect flow through to see if Taq is bound to the resin.

Optional: Load supernatant onto Ni column, wash with 20 mM Imidazole in lysis buffer (Buffer B), elute Taq with 300 mM imidazole in Buffer B. Desalt fractions containing Taq using gel filtration column. Combine fractions containing Taq and concentrate by spin concentrator.

22. Wash column with 6 CV of Buffer C, or until no protein or DNA is detected. Collect washes.

23. Elute Taq with Buffer D.

24. Pool fractions containing Taq protein, measure protein concentration. Run SDS gel to check purity.

25. Dilute the partially purified polymerase four-fold with Buffer E (to a final concentration of 50 mM NaCl). Load it into a Q Sepharose FF column. Collect flow through.

26. Wash column with 150 mL Buffer F to wash the column. Check the OD₂₆₀ (it should be less than 0.1) and OD₂₈₀ (it should be less than 0.1).

27. Elute Taq with Buffer G.

28. Collect eluate in 5 to 15 ml per fraction, Taq will come out after 40 mL. Check the OD₂₆₀ and OD₂₈₀ of each fraction. Save all the fractions that have OD₂₈₀ >0.1.

29. Combine the fractions with OD₂₈₀>0.1. Run SDS-PAGE gel to check purity.

30. Citraconylate  combined fractions

31. Dialyze citraconylated Taq for 12 h against 3~6 changes of 100 ml  Storage Buffer. The volume will decrease and the protein concentration should be increased to 10-20 mg/ml.

Buffer A

50 mM Tris-HCl, pH 7.9~8.0

1 mM EDTA.

Buffer B

20 mM Tris-HCl, pH 7.9~8.0

1 mM EDTA

0.5% Tween 20

0.5% Nonident-P40

Buffer C

Buffer B with 50 mM NaCl

or replace Tris-HCl with HEPES buffer. pH 7.9

Buffer D

Buffer B with 200 mM NaCl

Optional: if Taq peak is broad, increase NaCl to 500 mM.

Buffer E

20 mM HEPES (pH 8.3) or Tris-HCl, pH 8.0

Buffer F

20 mM HEPES (pH 8.0) or Tris-HCl, pH 7.9

50 mM KCl.

Buffer G

20 mM HEPES or Tris-HCl, pH 7.9 or pH 8.0

500 mM KCl.

Storage Buffer

20 mM HEPES, pH 7.9

100 mM KCl

0.1 mM EDTA

1 mM dithiothreitol

50% glycerol.