IGEM:British Columbia/Protocols/Making Plates or Liquid Media

Materials

• diH₂O
• Autoclavable container (1L glass bottles etc.)
• 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)
• LB Broth powder or LB Agar powder

Equipment

• Petri Dishes
• Pipet gun
• Disposable 10mL pipet
• Parafilm
• Tinfoil (if using Tetracycline)

Method

• Calculate amount of media needed (20mL/plate)
• Pour appropriate amount of diH₂O (~75% of final volume) into autoclavable container
• Add appropriate amount of LB Agar powder (37g/L of final volume) or LB Broth Powder (25g/L of final volume)
• Dissolve powder, then add diH₂O up to final volume
• Autoclave
• Cool to 55-60°C, but no lower
• 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/mL media
• Quickly pour or pipet into plates. Leave to dry 20-30 min before using (will dry faster if left at 37C). Alternatively, leave to solidify 20-30 min before spread plating with appropriate volume of antibiotic
• For storage, seal with parafilm and label according to the colour scheme(Red - Amp, Black- Chlor, Green - Tet, Blue - Kan) Keep at 4°C. If using tetracycline wrap in foil

• To make liquid media, skip steps 4, 7, 9-10. Store at 4°C, wrapping in foil if needed.