Editing Taq377 Purification (section)

== '''Purification Protocol 3'''  ==

1. Suspend 5 g sedimented cells in 25 ml lysis buffer (50 mM NaH2PO4 , 300 mM NaCl, 10 mM imidazole, 0.1 mM PMSF (phenylmethylsulfonyl fluoride), 1 mM DTT, pH 8.0)<br> 

2. lyse by freeze-thaw then ultrasound.<br> 

3. Add DNase I to a final concentration of 20 mg/ml and MgCl2 to a final concentration of 4 mM. <br> 

4. Incubate&nbsp; at 25° C. for 30 min. <br> 

5. Heat incubation for 30 min at 72° C.<br> 

6. Spin (22,000<span style="background-color: navy; color: white;" />g).<br> 

7. Collect supernatant.<br> 

8. Load to a 10 ml Ni-NTA superflow column (Qiagen). <br> 

9. Wash column&nbsp; with two volumes of buffer A (50 mM NaH2PO4 , 300 mM NaCl, 10 mM imidazole, pH 8.0). <br> 

10. Eluate Taq with a volume of 100 ml of a linear gradient, starting with buffer A and ending with buffer B (50 mM NaH2PO4 , 300 mM NaCl, 250 mM imidazole, pH 8.0). <br> 

11. The His-tagged Taq<span style="background-color: navy; color: white;" />279 is further purified by anion exchange chromatography with Q sepharose.<br> 

12. Dialyse the fraction containing the purified His-tagged Taq<span style="background-color: navy; color: white;" />279 polypeptide against storage buffer&nbsp;(50% [v/v] glycerol, 100 mM KCl, 20 mM Tris HCl pH 8.5, 0.1 mM EDTA, 1 mM DTT, 0.25% Thesit).<br> 

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1. 10X Taq Buffer with KCl<br>100mM Tris-HCl (pH 8.8 at 25<span style="background-color: navy; color: white;" />°C),<br>500mM KCl,<br>0.8% Nonidet P40. 

2. 10X Taq Buffer with KCl and 15mM MgCl2<br>100mM Tris-HCl (pH 8.8 at 25<span style="background-color: navy; color: white;" />°C),<br>500mM KCl,<br>0.8% Nonidet P40,<br>15mM MgCl2. 

3. 10X Taq Buffers with KCl Set<br>100mM Tris-HCl (pH 8.8 at 25<span style="background-color: navy; color: white;" />°C),<br>500mM KCl,<br>0.8% Nonidet P40,<br>MgCl2 different in each buffer (12.5-30mM) 

4. 10X Taq Buffer with (NH4)2SO4<br>750mM Tris-HCl (pH 8.8 at 25<span style="background-color: navy; color: white;" />°C),<br>200mM (NH4)2SO4,<br>0.1% Tween 20. 

5. 10X Taq Buffer with (NH4)2SO4 and 20mM MgCl2<br>750mM Tris-HCl (pH 8.8 at 25<span style="background-color: navy; color: white;" />°C),<br>200mM (NH4)2SO4,<br>0.1% Tween 20,<br>20mM MgCl2. 

6. 10X Taq Buffer without Detergent<br>100mM Tris-HCl (pH 8.8 at 25<span style="background-color: navy; color: white;" />°C),<br>500mM KCl. 

7. 10X Pfu Buffer with 20mM MgSO4<br>200mM Tris-HCl (pH 8.8 at 25<span style="background-color: navy; color: white;" />°C), 100mM (NH4)2SO4, 100mM KCl, 1% (v/v) Triton X-100, 1mg/ml BSA and 20mM MgSO4. 

8. 10X Pfu Buffer<br>200mM Tris-HCl (pH 8.8 at 25<span style="background-color: navy; color: white;" />°C), 100mM (NH4)2SO4, 100mM KCl, 1% (v/v) Triton X-100, 1mg/ml BSA<br> 

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