Difference between revisions of "Team:UiOslo Norway/Experiments/In vitro Assay Methanol Dehydrogenase"

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<p> coming soon </p>
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<a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments" >
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<h3> <i> in vitro </i> methanol dehydrogenase assay using E. coli raw extract
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<ul>
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  <li><p>Inoculate a preculture LB 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) and induce protein expression by adding IPTG to a final concentration of 1 mM at an OD600 of 0.4- 1.0</p></li>
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  <li><p>Incubate at 37 °C for 3 hours</p></li>
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  <li><p>Harvest the culture by centrifugation at 8000 x g for 5 minutes</p></li>
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  <li><p>Resuspend the cell pellet in 1 ml 50 mM K2HPO4 buffer (pH 7.4)</p></li>
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  <li><p>Add glass beads (0.1mm diameter) and disrupt the cells by shaking for 12 minutes</p></li>
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  <li><p>Centrifugation at 16000 x g for 5 minutes</p></li>
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  <li><p>Keep the soluble fraction for the assay</p></li>
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  <li><p>Perform the enzyme assay in a total volume of 1 ml</p></li>
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  <li><p>Add preheated 50 mM K2HPO4 buffer (pH 7.4) containing 5 mM MgSO4</p></li>
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  <li><p>Add 50 ul of soluble fraction</p></li>
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  <li><p>Add 1 M methanol</p></li>
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  <li><p>Add 200 uM NAD+</p></li>
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  <li><p>Detect the absorption at 340 nm</p></li>
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</ul>
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<h3> <i> in vitro </i> methanol dehydrogenase assay using purified NAD+ dependent methanol dehydrogenase
 +
<ul>
 +
  <li><p> </p></li>
 +
  <li><p>Perform the enzyme assay in a total volume of 1 ml</p></li>
 +
  <li><p>Add preheated 50 mM K2HPO4 buffer (pH 7.4) containing 5 mM MgSO4</p></li>
 +
  <li><p>Add 50 ul of soluble fraction</p></li>
 +
  <li><p>Add 1 M methanol</p></li>
 +
  <li><p>Add 500 uM NAD+</p></li>
 +
  <li><p>Detect the absorption at 340 nm</p></li>
 +
</ul>
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Revision as of 08:08, 9 September 2015

Back to Protocols


in vitro methanol dehydrogenase assay using E. coli raw extract

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

  • Inoculate a mainculture (50 ml) and induce protein expression by adding IPTG to a final concentration of 1 mM at an OD600 of 0.4- 1.0

  • Incubate at 37 °C for 3 hours

  • Harvest the culture by centrifugation at 8000 x g for 5 minutes

  • Resuspend the cell pellet in 1 ml 50 mM K2HPO4 buffer (pH 7.4)

  • Add glass beads (0.1mm diameter) and disrupt the cells by shaking for 12 minutes

  • Centrifugation at 16000 x g for 5 minutes

  • Keep the soluble fraction for the assay

  • Perform the enzyme assay in a total volume of 1 ml

  • Add preheated 50 mM K2HPO4 buffer (pH 7.4) containing 5 mM MgSO4

  • Add 50 ul of soluble fraction

  • Add 1 M methanol

  • Add 200 uM NAD+

  • Detect the absorption at 340 nm

in vitro methanol dehydrogenase assay using purified NAD+ dependent methanol dehydrogenase

  • Perform the enzyme assay in a total volume of 1 ml

  • Add preheated 50 mM K2HPO4 buffer (pH 7.4) containing 5 mM MgSO4

  • Add 50 ul of soluble fraction

  • Add 1 M methanol

  • Add 500 uM NAD+

  • Detect the absorption at 340 nm