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[Bio101] Preparation of Taq DNA Polymerase

Molecular Biology > DNA > PCR
Author: Wei Zheng
Wei ZhengAffiliation: Keck Biotech Services, Yale University, New Haven, USA
For correspondence: wei.zheng.madison@gmail.com
Bio-protocol author page: a10
10/5/2011, 9087 views, 1 Q&A

[Abstract]

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.


How to cite this protocol: (2011). Preparation of Taq DNA Polymerase. Bio-protocol Bio101: e136. http://www.bio-protocol.org/e136



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