Reference: Engelke, D. R. et al. Anal. Biochem. 191:396-400 (1990).
Pluthero, F. G. et al. NAR. 21:4850-4851 (1993).
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.
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)2SO4 / 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).
17) Titre by assaying serial dilutions cf comercial Taq.
18) Aliquot concentrated Taq polymerase and store at -20°C.
Terrific broth (TB); per litre
24gm yeast extract
4ml glycerol (autoclaved).
100ml 0.17M KH2PO4(2.31gm/100ml) / 0.72M K2HPO4 (12.54gm/100ml); Autoclaved separately.
Buffer A (Require 100ml)
50 mM Tris (pH 7.9)
Buffer B / 100ml (Require 1000ml)
20mM Hepes (pH7.9) 2ml (1M)
1mM EDTA 1ml (0.1M)
0.5% Tween-20 0.5ml
Buffer C / 500ml (Require 500ml)
20mM Hepes (pH 7.9) 10ml (1M)
1mM EDTA 5ml (0.1M)
0.5% Tween-20 2.5ml
Dialysis Buffer / 2L (Require 2000ml)
20mM Hepes (pH 7.9) 40ml (1M)
1mM EDTA 4ml (0.5M)
50% glycerol 1L
Dilution Buffer Required to dilute Taq
20mM HEPES (pH 7.9)
Dr. Ian Spendlove,
Academic Clinical Oncology,
Tel: +44 (0)115 9691169 ex47608,
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