Home-made Taq Polymerase Purification

 

Home-made Taq Polymerase Purification

I also have the bacteria containing the clone. It appears to produce lots of Taq and is quite stable.
The proceedure takes 4 days start to (15 000 units of Taq) finish.
The Taq also appears very stable and reliable. I made 15 mls a year ago and it still works fine.

Reference: Engelke, D. R. et al. Anal. Biochem. 191:396-400 (1990).
Pluthero, F. G. et al. NAR. 21:4850-4851 (1993).

Day 1

1) Inoculate two 2L flasks of TB/amp (500ml) with 15ml of an overnight of Taq bugs. These volumes may be scaled down.

2) Grow to an OD600 of 0.6 (approx mid log)

3) Add IPTG to 0.5mM (0.119gm/litre), grow o/n but not for more than 16hrs.

Day 2

N.B: All the following proceedures should be carried out on ice or at least 4°C.

4) Collect cells by centrifugation (3.5K / 15 mins / 4°C) and resuspend in 40ml buffer A.

5) Add an equal volume of buffer B (45-50ml) and incubate at 75°C for 1hr, with periodic mixing.

6) Centrifuge ( 8K / 15mins / 4°C).

7) Add 1.86mg of KCl / ml of supernatant.

8) Aliquot equal volumes of supernatant into each of 2 x 250ml centrifuge tubes (preferably conical) containing 75ml of washed Sigma DP-1 cation exchange resin (packed volume). This should be washed 2 x with sterile water and 4 x with ice cold Buffer B.

9) Vortex tubes well and incubate on a shaking platform (30mins / 4°C).

10) Centrifuge (approx 3K / 2min / 4°C) to pellet resin and discard supernatant.

11) Wash resin 4 x with 100-200ml of ice cold buffer B, remove supernatant by aspiration.

12) Elute 3 x with one packed bed volume of ice cold buffer C.

13) Add 30gm (NH₄ )₂ SO₄ / 100ml of eluate while stirring rapidly.

N.B: At this point it is of great advantage if you use conical or round bottomed tubes. i.e. 50ml tubes for the 8x50 rotor. Prior to this the sample may be handled in 250ml centrifuge bottles for ease of use.

14) Centrifuge at 12-15Krpm for 10mins at 4°C.

15) Resuspend pellet (weakly translucent) in 25-35ml of buffer C.

16) Dialise 2 x against 2L of dialysis buffer (6-18hrs / 4°C).

Day 3

17) Titre by assaying serial dilutions cf comercial Taq.

18) Aliquot concentrated Taq polymerase and store at -20°C.

Reagents

Terrific broth (TB); per litre
12gm tryptone
24gm yeast extract
4ml glycerol (autoclaved).
100ml 0.17M KH₂ PO₄ (2.31gm/100ml) / 0.72M K₂ HPO₄ (12.54gm/100ml); Autoclaved separately.

Buffer A (Require 100ml)
50 mM Tris (pH 7.9)
1mM EDTA
50mM Dextrose

Buffer B / 100ml (Require 1000ml)
20mM Hepes (pH7.9) 2ml (1M)
1mM EDTA 1ml (0.1M)
0.5% Tween-20 0.5ml
0.5%NP-40 0.5ml
0.5mM PMSF
50mM KCl

Buffer C / 500ml (Require 500ml)
20mM Hepes (pH 7.9) 10ml (1M)
1mM EDTA 5ml (0.1M)
0.5% Tween-20 2.5ml
0.5%NP-40 2.5ml
0.5mM PMSF
200mM KCl

Dialysis Buffer / 2L (Require 2000ml)
20mM Hepes (pH 7.9) 40ml (1M)
1mM EDTA 4ml (0.5M)
0.5mM PMSF
100mM KCl
50% glycerol 1L
1mM DTT

Dilution Buffer Required to dilute Taq
20mM HEPES (pH 7.9)
0.1mM EDTA
100mM KCl
50% glycerol