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Home-made Taq Polymerase Isolation
Author: 赖志兵, updated date: , view: 43, Q&A: 0

Procedure

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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