IGEM:PennState/2006/Growthmedia

Growth Media

LB

Media (per liter)

• 10 g Bactotryptone
• 5 g Yeast Extract
• 10 g NaCl
• pH to 7.0 with NaOH/HCl
• Autoclave

Plates

• Add 15 g Bacto-Agar per liter media
• Autoclave for 25 minutes
  • If antibiotics necessary, add only once media has cooled to ~45ºC, or is comfortable to the touch
• Pour in sterile environment (~15ml per plate)
• Cool in such a way as to avoid accumulation of condensation, while maintaining sterility
• Seal with parafilm, place in bag, and store at 4ºC

LBM

Media (per liter)

• 10 g Bactotryptone
• 5 g Yeast Extract
• 5 g NaCl
• 0.4066 g MgCl₂.6H₂O (or 2mL 1M MgCl₂)
• pH to 7.0 with NaOH/HCl
• Autoclave

Plates

• Add 15 g Bacto-Agar per liter media
• Autoclave for 25 minutes
  • If antibiotics necessary, add only once media has cooled to ~45ºC, or is comfortable to the touch
• Pour in sterile environment (~15ml per plate)
• Cool in such a way as to avoid accumulation of condensation, while maintaining sterility
• Seal with parafilm, place in bag, and store at 4ºC

Top Agar (per liter)

• 500 mL LBM
• 500 mL DI H₂O
• 8 g Bacto-Agar
• Autoclave

SOB

Media (per liter)

• 20 g bactotryptone
• 5 g bacto-yeast extract
• 0.5 g NaCl
• 0.19 g KCl
• pH to 7.0 with NaOH/HCl
• Autoclave for 25 minutes
• Add filter-sterilized MgCl₂ and MgSO₄ to yield final concentration of 10 mM each

Final Concentration:

• 0.5% Yeast Extract
• 2% Tryptone
• 10mM NaCl
• 2.5 mM KCl
• 10 mM MgCl2
• 10 mM MgSO4

SOC

Media

• Add filter-sterilized glucose to SOB for 20 mM final concentration of glucose

Minimal Media

M9 Salts (per liter)

• 1. Make M9 salts
• 2. To make M9 Salts aliquot 800ml H2O and add
  • 64g Na2HPO4-7H2O
  • 15g KH2PO4
  • 2.5g NaCl
  • 5.0g NH4Cl
  • Stir until dissolved
  • Adjust to 1000ml with distilled H2O
  • Sterilize by autoclaving

Media (per liter)

• 3. Measure ~700ml of distilled H2O (sterile)
• 4. Add 200ml of M9 salts
• 5. Add 2ml of 1M MgSO4 (sterile)
• 6. Add 20 ml of 20% glucose (or other carbon source)
• 7. Add 100ul of 1M CaCl2 (sterile)
• 8. Adjust to 1000ml with distilled H2O