Team:British Columbia/Notebook/Protocols/media

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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:

1. Pour appropriate amount of diH2O (400mL) into autoclavable container.

2. Add LB broth powder (Check amount on stock container).

3. Mix as well as you can by swirling and gentle shaking.

4. Autoclave.

• Mark bottle with autoclave tape, make sure the lid is loose*

• Follow directions on autoclave*

• Set to 30 min, turn timer counterclockwise* (CHECK LATER)

• Before removing from autoclave make sure the pressure is at 0*

• Use heat-resistant gloves to remove from Autoclave*

5. Cool until you can hold your wrist against the bottle for 5 seconds without pain.

6. 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.

7. Swirl gently to mix antibiotics.

8. 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.

Notes:

Ampicillin degrades quickly, so don't make plates more than 2-3 weeks in advance and never leave them at 37°C for more than 16 hours.

Kanamycin can last 2-3 months. I don't know about chloramphenicol and tetracycline (not that stable, use like Amp), but they seem to be quite stable as well. Tetracycline is light sensitive.