Crm at 18:02, 3 May 2017

2017-05-03T18:02:03Z

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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).

{| style="width: 500px" cellspacing="1" cellpadding="1" border="1"
|-
| 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.

[[File:105-1811 vPol QHP 3 1 2017.jpeg|RTENOTITLE]]

{| style="width: 500px" cellspacing="1" cellpadding="1" border="1"
|-
| 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.

{| style="width: 500px" cellspacing="1" cellpadding="1" border="1"
|-
| 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 
|}

[[File:Protein Expression SDS-PAGE 105-1811 vPol QHP 3 1 2017.jpg|RTENOTITLE]]

[[File:FLP vPol purification 9 2013.jpg]]

September, 2013 lane5-8 [http://excellgen.net/eln/index.php/1813_1811 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.
[[Category:My ELN]]

MS 2 22 2017 - Revision history

Crm at 18:02, 3 May 2017