MS 2 22 2017

1. Grow 1811 in LB/K/C

Expression

1. Take out 1811 glycerol stock and inoculate 200 ml LB//Kan/Chl.

2. Grow at 37 ºC o/n.

3. Add 1 ml of 10,000X Kan and 1 ml of 10,000X Chl, 20 ml of 20X50 solution to 1 L LB medium to make Autoinduce Medium

4. Split the overnight culture  into 8 L Autoinduce Medium, Grow 37 ºC  2~4 hours.

5. When cell density OD₆₀₀ is ~0.8, add 0.5 ml of 1 M IPTG to 1 L culture and induce at 37 ºC O/N. 

6. In the morning, spin down cells, combine. Weigh cell pellet, record weight is 44.50g. Store cell pellet at -20 ºC.

Purification

1. Add 800 ml Buffer A  to cell pellet, resuspend cell pellet using a spatula

2. Homogenize the cell suspension with a homogenizer.

3. Incubate the homogenate at 75 ^oC for 30 min, mix ocasionally, cool on ice for 15 min.

4. Spin at 6000 rpm for 15 min at 4 ^oC using high speed centrifuge, collect supernatant. 

5. Add cold 400 ml Buffer A to pellet. Homogenize again. 

6. Spin at 6000 rpm for 15 min at 4 ^oC using high speed centrifuge, collect supernatant. 

5. Transfer supernatant to ultracentrifuge tubes.  Cool ultraspeed rotor with dry ice for 5 min.

6. Spin at 25,000 rpm for 30 min at 4 ^oC using ultra speed centrifuge, collect clarified supernatant.

7. Load supernatant to 20 ml Ni column in cold room, flow rate ~ 1 ml/min

8. Wash Ni column with 200~500  ml Ni Washing Buffer (Buffer A).

9. Wash Ni column with 50 ml  20 mM Tris-HCl Buffer, pH 8.0

16. Elute Taq enzyme with ~200 ml Buffer B, Collect 5 ml fractions using a fraction collector.

17. Measure protein concentration in each fraction. Identify protein peak, combine fractions with A₂₈₀ >0.05 (2-9).

1	-0.06
2	-1.70
3	5.62
4	5.01
5	2.11
6	1.39
7	0.53
8	0.20
9	0.14
10	0.09
11	0.01
12	-0.01
13	-0.01
14	-0.02
15	-0.01
16	0.00
17	0.01
27	0.01
28	0.04

18.  Add 1 volume of cold Dilution Buffer.

19. Load Taq onto a  20~25 ml Q HP column (1.6 X10) using a peristaltic pump. With speed 15.

20. Use Dilution Buffer as blank, measure A₂₈₀ of sample, A₂₈₀=0.05.

21. Wash column with 100 ml Tris-HCl Buffer, flow rate 2 ml/min

22. Elute Taq using 200 ml of linear gradient of  

A: Q HP Washing Buffer

B: Q HP Elution Buffer  

Flow rate 1.5 or 2 ml/min. Use ddH₂O as blank, measure A₂₈₀.

20	0.14
21	9.54
22	14.45
23	2.69
24	1.91
25	0.76
26	0.65
27	0.53
28	0.37
29	0.32
30	0.32
31	0.11

23. Run SDS-PAGE gel on the first peak. If there is anothe peak at ~600~800 mM KCl, it could be DNA/RNA peak, discard the DNA/RNA peak.

Number	1	2	3	4	5	6	7	8	9	10	11	12
sample	MW	21	22	23	25	27	29	Taq 2/18	1032 2/1	95	104 1	104 2
uL	10	5	5	10	15	15	15	15	15	15	15	15
buffer uL		5	5	5	5	5	5	5	5	5	5	5

September, 2013 lane5-8 http://excellgen.net/eln/index.php/1813_1811

24. Combine fractions containing Taq (20-30).

25. Use ddH₂O wash the gel. Boil the gel in microwave for 2 min, two times.

26. Use N3-EF wash Buffer to boil the gel in microwave for 2 min.

27. Use ddH₂O wash the gel. boil the gel in microwave for 2 min, two times.

28. Dialysis. Add combined fractions into strip. Use Dialysis Buffer (Storage Buffer) to wash the strip.

Regeneration of Ni column

1. Wash Ni column with 5 CV of water.

2. Wash column with 3 CV of 100 mM EDTA, 500 mM NaCl.

3. Wash the column with 5 CV of H2O.

4. Recharge the column with 1 column volume of 100 mM NiSO4, or NiCl2.

5. Wash the column with 10 column volumes of H2O.

6. Equilibrate with 3 volumes of a suitable buffer (e.g., Buffer A).

Buffer A

20 mM Tris-HCl, pH 7.9~8.0

500 mM NaCl

0.1 mM EDTA

0.1% Tween 20

0.1% Nonident-P40

Ni Washing Buffer

20 mM Tris-HCl, pH 7.9~8.0

800 mM NaCl

10 mM imidazole 

Tris-HCl Buffer 

20 mM Tris-HCl, pH 7.9~8.0

Buffer B (Low Salt)

20 mM Tris-HCl, pH 7.9~8.0

100 mM NaCl

150 mM imidazole 

5% glycerol

Dilution Buffer

20 mM Tris-HCl, pH 7.9~8.0, optional: 5% glycerol

Q HP Washing Buffer

20 mM Tris-HCl, pH 7.9~8.0

50 mM KCl

5% Trehalose

Q HP Elution Buffer

20 mM Tris-HCl, pH 7.9~8.0

1 M KCl

5% Trehalose

Buffer E

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

Buffer F

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

50 mM KCl. 

Buffer G

20 mM HEPES or Tris-HCl, pH 7.9 or pH 8.0

500 mM KCl.

Storage Buffer

20 mM HEPES, pH 7.5

100 mM KCl

0.1 mM EDTA

1 mM dithiothreitol

50% glycerol.