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Home-made Taq Polymerase Isolation
Last updated date: Apr 1, 2024 Views: 3438
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Home-made Taq Polymerase Isolation Protocol
Protocol-As taken from Admud
Prepare 300ml Buffer A; 100ml Lysis Buffer; 1 Liter Storage Buffer; IPTG 125 mg; Lysozyme 200 mg; (NH4)2SO4 60 gram; 4x250ml tubes; 1Liter LB; 1ml 1MDTT;
1. Streak out to single colonies
2. Start an overnight culture
3. 500µl overnight culture into 1Liter LB (Ampicillin 50mg/L)
4. Grow at 37°C for 11 hours (OD600 is 0.8)
(0.5ml culture Taq/BL21 inoculated into 1 liter LB+Ampiicillin, 2 or 3 hours at 37°C to reach OD600= 0.2; add IPTG, then culture 16 to 20 hours)
5. Add IPTG to concentration of 125 mg/l (about 0.5mM)
6. Grow 10-12 hours (induction period)
7. Centrifuge and wash pellet in 100ml Buffer A/L culture
8. Centrifuge and resuspend in 50ml Buffer Buffer A +4mg/ml lysozyme (sigma L-6876-1g) and vortex to mix it, then split into four tubes
9. Incubate 15 min at Room Temperature.
10. Add equal volume lysis buffer
11. Incubate lysis mixture at 75°C for 1 hour.
12. Transfer lysis mixture to plastic bottles
13. Centrifuge 15,000 RPM for 10 minutes at 4°C. (10000RPMX15 min at 4°C)
14. Transfer clarified lysate to a new tube
15. Centrifuge 15,000 RPM for 10 minutes at 4°C. (10000RPMX15 min at 4°C) again to make the lysate clean.
16. Transfer clarified lysate to a strile pyrex beaker
17. Add 30 gram powdered (NH4)2SO4 per 100ml lysate (Stirring rapidly at Room Temperature)
18. Centrifuge 15,000RPM for 10 minutes. (10000RPMx 20 min at 4°C)
19. Harvest all protein precipitate and resuspend in 20 ml of Buffer C per 100ml of original cleared lysate
20. Dialyze against at least 2 changes of 12 hour each of storage buffer at 4°C. (each time 400ml dialysis buffer)
21. After dialysis, dilute 1:1 with storage buffer and store at -70°C.
Buffer A: | to make 1 liter | Prepare 300ml | |
50 mM Tris-HCL pH 7.9 | 50 ml 1M Tris-HCL pH 7.9 | 15ml | |
50mM Dextrose | 9 gram dextrose | 2.7g | |
1mM EDTA | 2ml 0.5M EDTA | 0.6ml | |
add water to 1 liter | 300ml | ||
Lysis Buffer | to make 1 liter | Prepare 100ml | |
10mM Tris-HCL pH 7.9 | 10 ml 1M Tris-HCL pH 7.9 | 1ml | |
50 mM KCl | 50 ml 1M KCl | 5ml | |
1mM EDTA | 2 ml 0.5M EDTA | 0.2ml | |
1mM PMSF | 10ml 0.1M PMSF | 1ml | |
0.5% Tween 20 | 5 gram Tweeen 20 | 0.5ml | |
0.5% Nonidet P 40 | 50 ml 10% Nonidet P 40 | 5ml | |
add water to 935 ml, then autoclave, just before using it, add PMSF, Tween20, and Nonidet P40 | Add water 93.5 ml, then autoclave, add PMSF, Tween20, and Nonidet P40 | ||
Buffer C / 25ml | |||
20mM Hepes (pH 7.9) | 0.5ml 1M Hepes (pH 7.9) | ||
50mM KCl | 1.25ml 1M KCl | ||
1mM EDTA | 0.05ml 0.5M EDTA | ||
0.5% Tween-20 | 0.125ml Tween-20 | ||
0.5mM PMSF | 0.125ml 100mM PMSF | ||
Add water to 24.75ml, then autoclave, add PMSF and Tween 20 just before use it. | |||
Storage Buffer | |||
50 mM Tris-HCL pH 7.9 | 50 ml 1M Tris-HCL pH 7.9 | ||
50 mM KCl | 50 ml 1M KCl | ||
0.1mM EDTA | 0.2 ml 0.5M EDTA | ||
1mM DTT | 1 ml 1M DTT | ||
0.5 mM PMSF | 5 ml 0.1 M PMSF | ||
50% glycenol | 500ml glyerin | ||
add water to 994 ml, then autoclave, add DTT and PMSF |
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