Team:British Columbia/Notebook/Protocols/media
From 2014.igem.org
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Liquid Media
Supplies:
- dH2O
- Autoclavable container (500mL Glass Bottles)
- Antibiotics (Stock solutions: Amp - 100mg/mL in 50% ethanol, Kan - 50mg/mL in H2O, Chl - 35mg/mL in 100%? ethanol, Tet - 15 mg/ml in 50% ethanol - all at -20°C)
- Tinfoil (if using Tet)
- LB broth powder
- Colour scheme: Amp - red, Kan - blue, Chl - black, Tet - green
Steps:
- Pour appropriate amount of diH2O (400mL) into autoclavable container.
- Add LB broth powder (Check amount on stock container).
- Mix as well as you can by swirling and gentle shaking.
- Autoclave:
- Mark bottle with autoclave tape, make sure the lid is loose.
- Follow directions on autoclave.
- Set to 30 min, turn timer counterclockwise, check back later.
- Before removing from autoclave make sure the pressure is at 0.
- Use heat-resistant gloves to remove from Autoclave.
- Cool until you can hold your wrist against the bottle for 5 seconds without pain.
- Add antibiotics (final concentrations: A-100-150μg/mL, K-50μg/mL, C-25μg/mL, Tet-10-15μg/mL); if using recommended stock solution concentrations, add 1μL of antibiotic stock per 1mL of media.
- Swirl gently to mix antibiotics.
- For storage, seal with parafilm and label according to the colour scheme. Keep at 4°C. If using a light-sensitive antibiotic, wrap in foil as well.