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

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<p> coming soon </p>
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<h1><i> In vivo </i> Methanol Dehydrogenase Assay </h1>
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<a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments" >
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Back to Protocols
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<p></br></p>
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<p>
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<ul>
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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 µl 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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<!---------------sponsors------------------------->
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<center><h1>iGEM UiOslo 2015 is sponsored by:</h1>
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<hr style="height:2px;border:none;color:black;background-color:black;" />
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<img src="https://static.igem.org/mediawiki/2015/a/ac/UiOslo_Statoil.jpg" Height="60px" hspace="15">
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<img src="https://static.igem.org/mediawiki/2015/e/e0/UiOslo_uio_logo.png" Height="60px" hspace="15">
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<img src="https://static.igem.org/mediawiki/2015/0/08/UiOslo_Enova1.jpg" Height="60px" hspace="15">
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<img src="https://static.igem.org/mediawiki/2015/e/eb/UiOslo_GATC-Biotech.jpg" Height="60px" hspace="15">
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<img src="https://static.igem.org/mediawiki/2015/4/4a/UiOslo_IDT.jpg" Height="60px" hspace="15">
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<img src="https://static.igem.org/mediawiki/2015/d/d9/UiOslo_Norwegian_Biochemical_Society.jpg" Height="60px" hspace="15">
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<img src="https://static.igem.org/mediawiki/2015/f/f0/UiOslo_SnapGene.jpg" Height="60px" hspace="15">
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</center>
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<hr style="height:2px;border:none;color:black;background-color:black;" />
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<!--------end sponsors---------------------------->
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</body>
 
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Latest revision as of 12:22, 18 September 2015

In vivo Methanol Dehydrogenase Assay

Back to Protocols


  • 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 µl 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

iGEM UiOslo 2015 is sponsored by: