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> | <li><p>Inoculate a preculture M9 minimal media containing the appropriate antibiotic and incubate at 37 °C overnight shaking at 220 rpm. </p></li> | ||
Revision as of 08:13, 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
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