IGEM:British Columbia/2009/Protocols/Media

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Contents

Making Plates/Liquid Media

Supplies Needed:

  • Petri dishes
  • 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.
    • Colour scheme: Amp - red, Kan - blue, Chl - black, Tet - green
  • Pipet gun + disposable 10mL pipet (optional)
  • Parafilm, tinfoil
  • LB broth powder, agar powder (OR LB Agar powder, OR bactotryptone/yeast extract/NaCl)

Steps:

  1. Calculate amount of media needed (~10mL/plate).
  2. Pour appropriate amount of diH2O (~75% of final volume) into autoclavable container.
  3. Add LB broth powder (20g/L of final volume).
  4. Add agar powder (10g/L of final volume).
  5. Dissolve powder, then add diH2O up to final volume.
  6. Autoclave.
  7. Cool to 55-60°C, but no lower.
  8. Add antibiotics (final concentrations: A-100-150μg/mL, K-50μg/mL, C-20μg/mL, Tet-10-15μg/mL); if using recommended stock solution concentrations, add 1μL/mL media.
  9. 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.
  10. 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.
  • To make liquid media, skip steps 4, 7, 9-10. Store at 4°C, wrapping in foil if needed.

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.

Antibiotic concentrations were taken from At the Bench, and in some cases differ slightly from recommended concentrations on OpenWetWare and elsewhere. If anyone has good reason for altering the concentrations up or down please feel free. (The colour scheme is also arbitrary, so someone with better colour coordination can change that too.)

If in a rush, don't worry about the volume change from adding powder and just pour your final volume straight off; it barely makes a difference.

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