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Team:British Columbia/Protocols/Biodesulfurization
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<h1>Detection of Biodesulfurization Intermediates by Reversed Phase HPLC</h1> | <h1>Detection of Biodesulfurization Intermediates by Reversed Phase HPLC</h1> | ||
#Grow overnight cultures of cells containing ''dsz'' operon in 5mL culture of LB. | #Grow overnight cultures of cells containing ''dsz'' operon in 5mL culture of LB. | ||
| - | #Measure O.D 600 with a spectrometer. Inoculate in | + | #Measure O.D 600 with a spectrometer. Inoculate in 200 mL culture of LB in a 500 mL flask such that the starting O.D 600 is 0.05 |
| - | #Incubate at 37°C shaking at | + | #Dibenzothiphene at 100ppm dissolved in 2% DMSO is added to the culture |
| + | #Incubate at 37°C shaking at 200 rpm. | ||
#Extract 25mL at O.D 600 intervals of 0.3, 0.7, and 1.0. | #Extract 25mL at O.D 600 intervals of 0.3, 0.7, and 1.0. | ||
| - | #Spin down cells using a centrifuge ( | + | #Spin down cells using a centrifuge (1600 g, 10 min) and extract 25mL of supernatant. |
#Acidify supernatant to pH of 2.0 with 6N HCl for efficient extraction of solutes. | #Acidify supernatant to pH of 2.0 with 6N HCl for efficient extraction of solutes. | ||
#Extract with equal volume of ethyl acetate. | #Extract with equal volume of ethyl acetate. | ||
Latest revision as of 04:03, 4 October 2012
Detection of Biodesulfurization Intermediates by Reversed Phase HPLC
- Grow overnight cultures of cells containing dsz operon in 5mL culture of LB.
- Measure O.D 600 with a spectrometer. Inoculate in 200 mL culture of LB in a 500 mL flask such that the starting O.D 600 is 0.05
- Dibenzothiphene at 100ppm dissolved in 2% DMSO is added to the culture
- Incubate at 37°C shaking at 200 rpm.
- Extract 25mL at O.D 600 intervals of 0.3, 0.7, and 1.0.
- Spin down cells using a centrifuge (1600 g, 10 min) and extract 25mL of supernatant.
- Acidify supernatant to pH of 2.0 with 6N HCl for efficient extraction of solutes.
- Extract with equal volume of ethyl acetate.
- Dry extract with nitrogen gas and re-suspend in mobile phase (80% acetonitrile).
- Filter the resuspended extract using a 0.45 μm PTFE or nylon filter.
- Run the sample on an HPLC using a C18 column (150 x 3 mm) and a flow rate of 0.8 ml/min. Monitor the sample at 280 nm.
