Preparation of Taq DNA Polymerase

Material

1.      Dialysis tubing cellulose membrane avg. flat width 33 mm (Sigma-Aldrich cat. D9652)

 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 500μl of overnight culture. Incubate at 37°C for about 11 hours with shaking. These cultures were grown to an OD₆₀₀ of approx. 0.8.

2.         8:00 P.M., add 62.5 mg of IPTG, incubate at 37°C for 12 hours 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 for 15 min.

4.         Add 25 ml of lysis buffer, mix well, transfer prep to Pyrex flask, and incubate at 75°C for 1 hour.

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 room temperature for 1/2 hour.

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 hours, one 12 hours and one 7 hours, 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.

Reagents and 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 D.D. H₂O, autoclave, store at room temperature.

2.         Lysis Buffer:

10 mM Tris - HCl pH 7.9                             1 ml of 1 M Tris-HCl pH7.9

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% Nonidet P40                                      0.5 ml NP40

q.s. to 100 ml with D.D. H₂O, autoclave, store at room temperature.

* 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.         Ammonium Sulfate:

Sigma, Cat. No. A4418.

5.         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.

6.         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                      

Bring to a final volume of 10 ml. Autoclave and aliquot into 1.5 ml tubes. Store at -20°C

7.         MgCl₂ solution:

Add 0.0238 g of MgCl₂ to 10 ml of D.D.H₂O.  Autoclave and divide into 1.5 ml tubes, store at -20°C

 Reference

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