Difference between revisions of "Team:UiOslo Norway/Experiments/In vivo Assay Methanol Dehydrogense"

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<h3> <i> in vivo </i> methanol dehydrogenase assay
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  <li><p>Inoculate a preculture M9 minimal media containing the appropriate antibiotic and incubate at 37 °C overnight shaking at 220 rpm. </p></li>
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  <li><p>Inoculate a mainculture (50 ml) with an OD600 of at least 0.3, add IPTG to a final concentration of 0.1 mM and grow the culture until the stationary phase is reached. </p></li>
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  <li><p>Measure OD600 of the culture and harvest a cell pellet of an OD600 of 1 in a volumne of 50 ml by centrifugation at 8000 x g for 10 minutes at roomtemperature</p></li>
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  <li><p>Resuspend the cell pellet in 50 ml M9 minimal media and measure the actual OD600</p></li>
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  <li><p>Start the experiment by adding 2 ml of 10 % (v/v) methanol</p></li>
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  <li><p>Directly after adding the methanol take a 600 ul sample</p></li>
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  <li><p>Take a 600 µl sample every 5 minutes over a 1 hour </p></li>
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  <li><p>Pellet the harvested cells by centrifugation at 11000 x g for 5 minutes at 4 °C</p></li>
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  <li><p>Use 500 µl of the supernatant with 500 µl of Nash reagent and measure absorption at 412 nm and determine the concentration of formaldehyde</p></li>
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Revision as of 08:12, 9 September 2015

in vivo methanol dehydrogenase assay

  • Inoculate a preculture M9 minimal media containing the appropriate antibiotic and incubate at 37 °C overnight shaking at 220 rpm.

  • Inoculate a mainculture (50 ml) with an OD600 of at least 0.3, add IPTG to a final concentration of 0.1 mM and grow the culture until the stationary phase is reached.

  • Measure OD600 of the culture and harvest a cell pellet of an OD600 of 1 in a volumne of 50 ml by centrifugation at 8000 x g for 10 minutes at roomtemperature

  • Resuspend the cell pellet in 50 ml M9 minimal media and measure the actual OD600

  • Start the experiment by adding 2 ml of 10 % (v/v) methanol

  • Directly after adding the methanol take a 600 ul sample

  • Take a 600 µl sample every 5 minutes over a 1 hour

  • Pellet the harvested cells by centrifugation at 11000 x g for 5 minutes at 4 °C

  • Use 500 µl of the supernatant with 500 µl of Nash reagent and measure absorption at 412 nm and determine the concentration of formaldehyde