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Team:Washington/Protocols/EG Assay
From 2012.igem.org
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<li> 2.8mL of filter sterilized 99.99% ethylene glycol</li> | <li> 2.8mL of filter sterilized 99.99% ethylene glycol</li> | ||
<li> 800mL of sterile double distilled water</li></ul> | <li> 800mL of sterile double distilled water</li></ul> | ||
| - | <li> Autoclave | + | <li> Autoclave the closed system of tubing, syringes, culture vessel, and empty bottle </li> |
| - | <ul><li> Wrap all open ends of the system with aluminium foil before autoclaving to prevent autoclave water from entering the closed tubing system</li></ul> | + | <ul><li> Wrap all open ends of the system with aluminium foil before autoclaving to prevent autoclave water from entering the closed tubing system </li></ul> |
<li> Place closed system into the turbidostat apparatus</li> | <li> Place closed system into the turbidostat apparatus</li> | ||
| - | <li> Carefully and sterilely decant previously made sterile M9 | + | <li> Carefully and sterilely decant previously made sterile M9 30mM ethylene glycol media into the media bottle </li> |
| - | <li> Place turbidostat into a 37º C incubator</li> | + | <li> Place the turbidostat into a 37º C incubator</li> |
| - | <li> Connect turbidostat to a computer with the turbidostat software and related libraries installed</li> | + | <li> Connect the turbidostat to a computer with the turbidostat software and related libraries installed</li> |
| - | <li> Start up turbidostat | + | <li> Start up turbidostat software program </li> |
<ul><li>Be sure to close incubator door to reduce amount of ambient light</li></ul> | <ul><li>Be sure to close incubator door to reduce amount of ambient light</li></ul> | ||
<li> Wait till program reads out optical density values - should see "0.000" as first optical density value</li> | <li> Wait till program reads out optical density values - should see "0.000" as first optical density value</li> | ||
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<li> Wait 12-24 hours </li> | <li> Wait 12-24 hours </li> | ||
<li> Stop turbidostat program </li> | <li> Stop turbidostat program </li> | ||
| - | <li> Retrieve data files from program folder and analyze them using your favorite mathematical software (ie: | + | <li> Retrieve data files from program folder and analyze them using your favorite mathematical software (ie: <a href=http://www.mathworks.com/products/matlab/><font size="2">Matlab</font></a> or <a href=http://www.wolfram.com/mathematica/><font size="2">Mathematica</font></a>)</li> |
</ol> | </ol> | ||
</ol> | </ol> | ||
</html> | </html> | ||
Latest revision as of 02:04, 4 October 2012
Turbidostat: Ethylene Glycol Assay
- Turbidostat Preparation
- Created M9 30mM ethylene glycol media
- Sterile 200mL of 5X M9 salts
- 2.8mL of filter sterilized 99.99% ethylene glycol
- 800mL of sterile double distilled water
- Autoclave the closed system of tubing, syringes, culture vessel, and empty bottle
- Wrap all open ends of the system with aluminium foil before autoclaving to prevent autoclave water from entering the closed tubing system
- Place closed system into the turbidostat apparatus
- Carefully and sterilely decant previously made sterile M9 30mM ethylene glycol media into the media bottle
- Place the turbidostat into a 37º C incubator
- Connect the turbidostat to a computer with the turbidostat software and related libraries installed
- Start up turbidostat software program
- Be sure to close incubator door to reduce amount of ambient light
- Wait till program reads out optical density values - should see "0.000" as first optical density value
- Cell Preparation
- Culture an overnight of MG1655 transformed with fucO pGA3K3 and aldA pGA1C3 in 2mL of TB with kanamycin and chloramphenicol
- Take 1mL from the overnight culture and pipette it into a 1.5mL microcentrifuge tube
- Pellet the 1mL aliquot at 4000g for 3 minutes
- Pour out the supernatant
- Resuspend the pelleted cells with 1mL of sterile PBS
- Repeat steps 3-5 two more times
- Turbidostat Inoculation and Operation
- Take a hypodermic needle attached to a 2.5mL syringe and aspirate 0.5mL of washed cells
- Open incubator and through the soft stopper at the top of the culture vessel, inject the cells into the culture vessel to an OD ~0.2
- Close incubator door
- Wait 12-24 hours
- Stop turbidostat program
- Retrieve data files from program folder and analyze them using your favorite mathematical software (ie: Matlab or Mathematica)
