Team:British Columbia/Protocols/Biodesulfurization

From 2012.igem.org

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<h1>Biodesulfurization with HPLC</h1>
<h1>Biodesulfurization with HPLC</h1>
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1. Grow overnight cultures of cells containing dsz operon in 5mL culture of LB.  
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#Grow overnight cultures of cells containing dsz operon in 5mL culture of LB.  
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#Measure O.D 600 with a spectrometer. Inoculate in 200mL culture of LB in a 500mL flask such that the starting O.D 600 is 0.05.  
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2. Measure O.D 600 with a spectrometer. Inoculate in 200mL culture of LB in a 500mL flask such that the starting O.D 600 is 0.05.  
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#Incubate at 37°C shaking at 200rpm.
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#Extract 25mL at O.D 600 intervals of 0.3, 0.7, and 1.0.
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3. Incubate at 37°C shaking at 200rpm.
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#Spin down cells using a centrifuge (1600g, 10min) and extract 25mL of supernatant.
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#Acidify supernatant to pH of 2.0 with 6N HCl for efficient extraction of solutes.
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4. Extract 25mL at O.D 600 intervals of 0.3, 0.7, and 1.0.
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#Extract with equal volume of ethyl acetate.
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#Dry extract with nitrogen gas and re-suspend in mobile phase (80% acetonitrile).  
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5. Spin down cells using a centrifuge (1600g, 10min) and extract 25mL of supernatant.
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#Filter the resuspended extract using a 0.45 μm PTFE or nylon filter.  
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#Run the sample on and HPLC using a C18 column (150 x 3 mm)and a flow rate of 0.8 ml/min. Monitor the sample at 280 nm.
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6. Acidify supernatant to pH of 2.0 with 6N HCl to inactivate enzymes.
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7. Extract with equal volume of ethyl acetate.
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8. Dry extract with nitrogen gas and re-suspend in mobile phase with 80% acetonitrile.  
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9. Filter extract resuspended in acetonitrile using reverse phase chromatography and C-18 column.  
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10. Elute mobile phase at a flow rate of 0.8 ml/min and peaks of DBT were detected at 280nm by HPLC.
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Revision as of 01:13, 4 October 2012

British Columbia - 2012.igem.org
UBC iGEM 2012 protocols


Biodesulfurization with HPLC

  1. Grow overnight cultures of cells containing dsz operon in 5mL culture of LB.
  2. Measure O.D 600 with a spectrometer. Inoculate in 200mL culture of LB in a 500mL flask such that the starting O.D 600 is 0.05.
  3. Incubate at 37°C shaking at 200rpm.
  4. Extract 25mL at O.D 600 intervals of 0.3, 0.7, and 1.0.
  5. Spin down cells using a centrifuge (1600g, 10min) and extract 25mL of supernatant.
  6. Acidify supernatant to pH of 2.0 with 6N HCl for efficient extraction of solutes.
  7. Extract with equal volume of ethyl acetate.
  8. Dry extract with nitrogen gas and re-suspend in mobile phase (80% acetonitrile).
  9. Filter the resuspended extract using a 0.45 μm PTFE or nylon filter.
  10. Run the sample on and HPLC using a C18 column (150 x 3 mm)and a flow rate of 0.8 ml/min. Monitor the sample at 280 nm.
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