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Preparation and Purification of Taq Polymerase
Preparation and Purification of Taq Polymerase
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).
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.
3. Add an equal volume of Buffer B and incubate at 75°C for 1 hr (mix the tube
periodically).
7. Add an equal volume of sterile ddH2O, mix well, and centrifuge at approximately
3,000 X g to pellet the resin.
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.
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).
17. Centrifuge to pellet the resin at approximately 3,000 X g for 2 min at 4°C.
20. Centrifuge to pellet the resin at approximately 3,000 X g for 2 min at 4°C.
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.
26. Repeat the elution of protein from the resin two more times (Steps #B23 to #B25).
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.
Solutions
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
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
BioReagents and Chemicals
Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Isopropyl beta-D-thiogalactopyranoside
HEPES
Potassium Phosphate, Dibasic
Potassium Chloride
Dextrose
Potassium Phosphate, Monobasic
Yeast Extract
Tryptone
Ampicillin
NP-40
DTT
Glycerol
EDTA
Ammonium Sulfate
Tween-20
PMSF
Tris
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.
4. It is advantageous to aliquot the supernatant collected from the resin into 50 ml conical
or round-bottom centrifuge tubes.