Editing MS 2 22 2017

1. Grow 1811 in LB/K/C

== <span class="mw-headline" id="Expression">Expression</span> ==

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

2. Grow at 37&nbsp;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&nbsp;'''Autoinduce Medium'''

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

5. When cell density OD<sub>600</sub> is ~0.8, add 0.5 ml of 1 M IPTG to 1 L culture and induce at 37 ºC&nbsp;O/N.&nbsp;

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



== <span class="mw-headline" id="Purification">Purification</span> ==

1. Add 800&nbsp;ml&nbsp;'''Buffer A'''&nbsp; to cell pellet, resuspend cell pellet&nbsp;using a spatula

2. Homogenize the cell suspension with a homogenizer.

3. Incubate the homogenate&nbsp;at 75&nbsp;<sup style="line-height: 1">o</sup>C for 30 min, mix ocasionally, cool on ice for 15 min.

4. Spin at 6000 rpm for 15&nbsp;min at 4&nbsp;<sup style="line-height: 1">o</sup>C using high speed centrifuge, collect supernatant.&nbsp;

5. Add cold 400 ml&nbsp;'''Buffer A'''&nbsp;to pellet. Homogenize again.&nbsp;

6. Spin at 6000 rpm for 15&nbsp;min at 4&nbsp;<sup style="line-height: 1">o</sup>C using high speed centrifuge, collect supernatant.&nbsp;

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

6. Spin at 25,000 rpm for 30 min at 4&nbsp;<sup style="line-height: 1">o</sup>C 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&nbsp;Ni column with 200~500 &nbsp;ml&nbsp;'''Ni Washing Buffer (Buffer A).'''

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

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

17. Measure protein concentration in each fraction. Identify protein peak, combine fractions&nbsp;with A<sub>280</sub> >0.05 (2-9).

{| style="width: 500px" cellspacing="1" cellpadding="1" border="1"
|-
| 1<br/>
| -0.06<br/>
|-
| 2<br/>
| -1.70<br/>
|-
| 3<br/>
| 5.62<br/>
|-
| 4<br/>
| 5.01<br/>
|-
| 5<br/>
| 2.11<br/>
|-
| 6<br/>
| 1.39<br/>
|-
| 7<br/>
| 0.53<br/>
|-
| 8<br/>
| 0.20<br/>
|-
| 9<br/>
| 0.14<br/>
|-
| 10<br/>
| 0.09<br/>
|-
| 11<br/>
| 0.01<br/>
|-
| 12<br/>
| -0.01<br/>
|-
| 13<br/>
| -0.01<br/>
|-
| 14<br/>
| -0.02<br/>
|-
| 15<br/>
| -0.01<br/>
|-
| 16<br/>
| 0.00<br/>
|-
| 17<br/>
| 0.01<br/>
|-
| 27<br/>
| 0.01<br/>
|-
| 28<br/>
| 0.04<br/>
|}

18. &nbsp;Add 1 volume of&nbsp;cold '''Dilution Buffer.'''

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

20. Use Dilution Buffer as blank, measure A<sub>280</sub> of sample, A<sub>280</sub>=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 &nbsp;

'''A:''&nbsp;'''''<b>Q HP Washing Buffer</b>

'''B: Q HP Elution&nbsp;Buffer&nbsp;'''&nbsp;

Flow rate 1.5 or 2 ml/min. Use ddH<sub>2</sub>O as blank, measure A<sub>280</sub>.

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

{| style="width: 500px" cellspacing="1" cellpadding="1" border="1"
|-
| 20<br/>
| 0.14<br/>
|-
| 21<br/>
| 9.54<br/>
|-
| 22<br/>
| 14.45<br/>
|-
| 23<br/>
| 2.69<br/>
|-
| 24<br/>
| 1.91<br/>
|-
| 25<br/>
| 0.76<br/>
|-
| 26<br/>
| 0.65<br/>
|-
| 27<br/>
| 0.53<br/>
|-
| 28<br/>
| 0.37<br/>
|-
| 29<br/>
| 0.32<br/>
|-
| 30<br/>
| 0.32<br/>
|-
| 31<br/>
| 0.11<br/>
|}

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

[[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&nbsp;[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 ddH<sub>2</sub>O 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 ddH<sub>2</sub>O 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.



== <span class="mw-headline" id="Regeneration_of_Ni_column">Regeneration of Ni column</span> ==

1. Wash Ni&nbsp;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.,&nbsp;''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

<br/>'''Ni Washing Buffer'''

20 mM Tris-HCl, pH 7.9~8.0

800 mM NaCl

10 mM imidazole&nbsp;



'''Tris-HCl Buffer&nbsp;'''

20 mM Tris-HCl, pH 7.9~8.0



'''Buffer B&nbsp;(Low Salt)'''

20 mM Tris-HCl, pH 7.9~8.0

'''100''' mM NaCl

150&nbsp;mM imidazole&nbsp;

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&nbsp;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&nbsp;



'''Buffer F'''

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

50 mM KCl.&nbsp;



'''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]]