Editing Wild-type Taq Purification

1. Transform '''BL21 (DE3) pLysS''' with FastTaq (pET30b-sso7d-Taq_d289, 377) plasmid. 

2. Grow on LB/Kan/Chl plate o/n.<br> 

3.&nbsp; Pick a colony, grow in 200 ml LB/Kan/Chl. 

'''''Optional:''''' screen colonies for high yield expression. 

4. Add 30 ml bacteria to 1 L Autoinduce Medium, Grow total of 6 L in 6 flasks at 30 <sup>o</sup>C or 37 <sup>o</sup>C O/N. <br> 

5. In the morning, spin down cells, wash cells with '''Buffer A''' or DI-water, spin. Weigh cell pellet, record weight.<br> 

6. Continue to grow cells for a few more days until total cell pellet is 100 g.<br> 

7. Add '''25''' ml&nbsp;'''Buffer B'''&nbsp; to cell pellet from 1 L culture, suspend cells by votexing.<br> 

8. Freeze cell pellet with dry ice.<br> 

9. Thaw cell pellet at 75 <sup>o</sup>C (water bath).<br> 

10. Repeat freeze-thaw for 1 or 2 more cycles.<br> 

11. Sonicate lysate if it is still sticky.<br> 

12. "Boil" lysate at 75 <sup>o</sup>C for 30 min, cool on ice for 15 min.<br> 

13. Spin at 12000 rpm for 10 min at 4 <sup>o</sup>C (Sorvall high speed centrifuge), collect supernatant.<br> 

14. Transfer supernatant to ultracentrifuge tubes. '''Balance to 0.1 g.'''<br> 

15. Spin at 25000 rpm for 10 min at 4 <sup>o</sup>C, collect clarified supernatant <strike>into a clean 500 ml centrifuge tube</strike>.<br> 

<strike>16. Add ~30 g ammonium sulfate (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> '''slowly''' into the '''100 ml '''of supernatant while stirring or mixing at room temperature.<br></strike> 

<strike>17. Incubate at room temperature for 15 min.<br></strike> 

<strike>18. Spin at 12000 rpm for 15 min at 4 <sup>o</sup>C.<br></strike> 

<strike>19. Resuspend pellet in 5 ml '''Buffer A''', wash centrifuge tube with up to 2 to 4 more times with '''Buffer A''', combine washes. Try not to form bubbles.<br></strike> 

<strike>20. Add saturated sterile (autoclaved) dibasic potassium phosphate, K<sub>2</sub>HPO<sub>4</sub>, (pH 10.3) to&nbsp; Taq suspension to final concentration of 10%, i.e., add 1 ml to 9 ml of suspension. Mix well for several seconds. Then add an equal volume of ethanol to the 10% salt solution and mix vigorously for several seconds. Spin for 2 min. Collect the top (organic ethanol) phase containing the Taq.</strike> 

<strike>Check the bottom (aqueous) phase for both Taq and nucleic acids by PCR and agarose gel.</strike> 

<strike><br></strike> 

<strike>At this step, the Taq in aqueous phase should be at least&nbsp; 1Ã10<sup>6</sup> units from 1 L culture. (i.e., 1 unit/5 ng protein. --&gt;1 million units/5 mg protein).<br></strike> 

<strike><br></strike> 

<strike>'''''Optional:''''' Before aquous two phase extraction, desalt Taq suspension with '''Buffer B''' by gel filtration (FPLC), Combined desalted fractions containing active Taq DNA polymerase.</strike>''<br>'' 

<br> 

<strike>21. Dilute Taq with '''Buffer C''' to 20% ethanol (1 ml Taq in ethanol + 4 ml '''Buffer C''').&nbsp;</strike> 

21. Load Taq onto a 150-ml BioRex 70 (Bio-Rad) ion exchanger column, pre-equilibrated with '''Buffer C'''. <u>'''Collect flow through''' </u>to see if Taq is bound to the resin. <br> 

''

'''''Optional:''''' Load supernatant onto Ni column, wash with 20 mM Imidazole in lysis buffer (Buffer B), elute Taq with 300 mM imidazole in Buffer B. Desalt fractions containing Taq using gel filtration column. Combine fractions containing Taq and concentrate by spin concentrator.<br>



22.&nbsp;Wash column with '''6''' CV of '''Buffer C''', or until no protein or DNA is detected. Collect washes.<br> 

23. Elute Taq with '''Buffer D'''.<br> 

24. Pool fractions containing Taq protein, measure protein concentration. Run SDS gel to check purity.<br> 

25. Dilute the partially purified polymerase four-fold with '''Buffer E '''(to a final concentration of 50 mM NaCl). Load it into a Q Sepharose FF column. Collect flow through.<br> 

26. Wash column with 150 mL '''Buffer F''' to wash the column. Check the OD<sub>260</sub> (it should be less than 0.1) and OD<sub>280</sub> (it should be less than 0.1). 

27. Elute Taq with '''Buffer G'''. 

28. Collect eluate in 5 to 15 ml per fraction, Taq will come out after 40 mL. Check the OD<sub>260</sub> and OD<sub>280</sub> of each fraction. Save all the fractions that have OD<sub>280</sub> &gt;0.1. 

29. Combine the fractions with OD<sub>280</sub>&gt;0.1. Run SDS-PAGE gel to check purity. <br> 

30. [[Taq purification|Citraconylate&nbsp;]] combined fractions<br> 

31. Dialyze citraconylated Taq for 12 h against 3~6 changes of 100 ml&nbsp; '''Storage Buffer. '''The volume will decrease and the protein concentration should be increased to 10-20 mg/ml. <br> 

<br> 

<br> 

'''Buffer A'''<br> 

50 mM Tris-HCl, pH 7.9~8.0<br> 

1 mM EDTA.<br> <br> 

'''Buffer B''' 

20 mM Tris-HCl, pH 7.9~8.0<br> 

1 mM EDTA<br> 

0.5% Tween 20<br> 

0.5% Nonident-P40<br> <br> 

'''Buffer C'''<br> 

Buffer B with 50 mM NaCl<br> 

or replace Tris-HCl with HEPES buffer. pH 7.9<br> 

<br> 

'''Buffer D'''<br> 

Buffer B with 200 mM NaCl<br> 

Optional: if Taq peak is broad, increase NaCl to 500 mM.<br> 

<br> 

'''Buffer E'''<br> 

20 mM HEPES (pH 8.3) or Tris-HCl, pH 8.0 <br> 

<br> 

'''Buffer F''' 

20 mM HEPES (pH 8.0) or Tris-HCl, pH 7.9 <br> 

50 mM KCl. <br> 

<br> 

'''Buffer G'''<br> 

20 mM HEPES or Tris-HCl, pH 7.9 or pH 8.0<br> 

500 mM KCl. <br> 

'''Storage Buffer'''<br> 

20 mM HEPES, pH 7.9<br> 

100 mM KCl<br> 

0.1 mM EDTA<br> 

1 mM dithiothreitol<br> 

50% glycerol.<br><br> 

[[Category:Protocols]]