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

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

Final Concentration:

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

2. Autoclave on slow exhaust for 25 min.

3. Add filter-sterilized MgCl2, MgSO4 solution to give final Mg2+ conc. of 20 mM (i.e. 10 mM MgCl2, 10 mM MgSO4)

SOC MEDIA SOB + 20 mM (final) glucose (aka dextrose)

BAD MEDIA (unkown?? M9??)

(SUPPLEMENTED W/ 0.1% YE) Final concentrations:

• 1X M9 salts (autoclaved)
• 1 mM MgSO4 (autoclaved)
• 0.16% glycerol (autoclaved)
• 0.148 μM thiamine/vitamin B1 (filter sterilized)
• 0.1% casamino acids (filter sterilized)
• 0.1% Yeast Extract (autoclaved)

Procedure:

1. These solutions should already be made, so add together w/sterile technique:

• 200 mL 5X M9 salts
• 1 mL 1 M MgSO4
• 2.27 mL 70% glycerol
• 50 μL thiamine (10mg/mL)
• 5 mL casamino acids (200 mg/mL)
• 10 mL 0.1g/mL Yeast Extract
• 782 mL sterile dH2O .
• 1 L M9 media (for plates just add 15 g bacto agar to recipe)

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