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

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<h1><i> In vitro </i> Methanol Dehydrogenase assay using <i>E. coli </i> raw extract
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<h1><i> In vitro </i> Methanol Dehydrogenase assay using <i>E. coli </i> raw extract</h1>
 
<a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments" >  
 
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   <li><p>Detect the absorption at 340 nm</p></li>
 
   <li><p>Detect the absorption at 340 nm</p></li>
 
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<h3> <i> in vitro </i> methanol dehydrogenase assay using purified NAD+ dependent methanol dehydrogenase
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<h1> <i> In vitro </i> Methanol Dehydrogenase assay using purified NAD+ dependent Methanol Dehydrogenase</h1>
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<a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments" >
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Revision as of 08:22, 9 September 2015

In vitro Methanol Dehydrogenase assay using E. coli raw extract

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  • 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 µl of soluble fraction

  • Add 1 M methanol

  • Add 200 µM NAD+

  • Detect the absorption at 340 nm

In vitro Methanol Dehydrogenase assay using purified NAD+ dependent Methanol Dehydrogenase

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  • 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