[Bio101] Preparation of Taq DNA Polymerase

Materials and Reagents

1. Dialysis tubing cellulose membrane avg. flat width 33 mm (Sigma-Aldrich, catalog number: D9652 )
2. E.coli BL21 strain 
3. Ammonium sulfate (Sigma-Aldrich, catalog number: A4418 )
4. IPTG
5. Ampicillin
6. (NH₄)₂SO₄
7. 1 M dextrose
8. 1 M Tris - HCl (pH 7.9)
9. 0.5 M EDTA (pH 8.0)
10. KCl
11. PMSF
12. Tween 20
13. NP40
14. Lysozyme
15. Glycerol
16. Triton-X 100
17. Lysis buffer (see Recipes)
18. Buffer A (see Recipes)
19. Pre - lysis buffer (see Recipes)
20. Storage buffer (see Recipes)
21. TAQ buffer (see Recipes)
22. MgCl₂ solution (see Recipes)
    

Equipment

1. 2 L flask
2. Centrifuges
3. Oakridge tubes
   

Procedure

Day1

1. Inoculate a single colony of E.coli BL21 strain with Taq plasmid driven by IPTG inducible promoter into 5 ml of LB broth containing 20 μl of ampicillin (100 mg/ml). Incubate at 37 °C overnight with shaking.

Day 2

1. 7:00 A.M. prepare 500 ml of LB broth containing 40 mg of ampicillin in a 2 L flask, add 500 μl of overnight culture. Incubate at 37 °C for about 11 h with shaking. Grow these cultures to an OD₆₀₀ of approx. 0.8.
2. 8:00 P.M., add 62.5 mg of IPTG, incubate at 37 °C for 12 h with shaking.
   

Day 3

1. Transfer overnight culture to 500 ml plastic bottle, centrifuge at 4.5 K for 10 min at 4 °C. Decant supernatant, and resuspend the prep in 50 ml of Buffer A.
2. Centrifuge at 4.5 K for 10 min at 4 °C, decant supernate, and resuspend the prep in 25 ml of pre-lysis buffer.
3. Incubate at room temperature (RT) for 15 min.
4. Add 25 ml of lysis buffer, mix well, transfer prep to Pyrex flask, and incubate at 75 °C for 1 h.
5. Divide the prep into two Oakridge tubes, cetrifuge at 15,000 rpm for 10 min at 4 °C.
6. Transfer supernate to clean flask, add 15 g of (NH₄)₂SO₄, stir rapidly at RT for 1/2 h.
7. Centrifuge at 15,000 rpm for 10 min at 4 °C.
8. Decant supernate, resuspend prep in 10 ml of Buffer A.
   

Day 3 & 4

1. The resuspended protein was dialyzed against 3 changes of storage buffer at 4 °C. (One 7 h, one 12 h and one 7 h, each with> 800 ml of storage buffer. The volume of the protein was reduced to about 5 ml after the dialysis.)
2. Protein was diluted 1:1 with sterilized storage buffer, and store at -80 °C.
   

Recipes

1. Buffer A
   50 mM Tris - HCl (pH 7.9)         25 ml of 1 M Tris - HCl (pH 7.9)
   50 mM dextrose                        25 ml of 1 M dextrose
   1 mM EDTA (pH 8.0)                1 ml of 0.5 M EDTA (pH 8.0)
   q.s. to 500 ml with ddH₂O, autoclave, store at RT.
   
2. Lysis buffer
   10 mM Tris - HCl (pH 7.9)        1 ml of 1 M Tris-HCl (pH 7.9)
   50 mM KCl                               5 ml of 1 M KCl
   1 mM EDTA (pH 8.0)                0.2 ml of 0.5 M EDTA (pH 8.0)
   1 mM PMSF*                            2 ml of 50 mM PMSF
   0.5% Tween 20                       0.5 ml Tween 20
   0.5% NP40                             0.5 ml NP40
   q.s. to 100 ml with ddH₂O, autoclave, store at RT.
   * Toxic material, always wear a mask and gloves when dealing with it!
   PMSF can only be dissolved in 100% EtOH.
   
3. Pre - lysis buffer
   Buffer A plus 4 mg/ml lysozyme.
   
4. Storage buffer
   50 mM Tris - HCI (pH 7.9)         150 ml of 1 M Tris- HCI (pH 7.9)
   50 mM KCl                                150 ml of 1 M KCl
   0.1 mM EDTA (pH 8.0)              0.6 ml of 0.5 M EDTA (pH 8.0)
   1 mM DTT                                 3 ml of 1 M DTT
   0.5 mM PMSF*                          30 ml of 0.5 mM PMSF
   50% glycerol                            1.5 L of glycerol
   q.s. to 3 L w/ D.D. H₂O, autoclave, store at RT.
5. TAQ buffer
   500 mM KCl                        5 ml of 1 M KCl
   100 mM Tris                        1 ml of of 1 M Tris (pH 9.0)
   1% Triton                           100 μl of Triton-X 100
   Bring to a final volume of 10 ml. Autoclave and aliquot into 1.5 ml tubes. Store at -20 °C.
6. MgCl₂ solution
   Add 0.0238 g of MgCl₂ to 10 ml of ddH₂O. Autoclave and divide into 1.5 ml tubes, store at -20 °C.
   

References

1. Pluthero, F. G. (1993). Rapid purification of high-activity Taq DNA polymerase. Nucleic Acids Res 21(20): 4850-4851.