IGEM:PennState/2006/Growthmedia

Growth Media and plates

LB Media

(Per L)

• 10 g bactotryptone
• 5 g yeast extract
• 10 g NaCl
• pH to 7.0 (~40uL 2M NaOH / L)

M9 Minimal Media

(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)

SOB Media

Final concentrations

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

Procedure

1. To 1 L, add:

• 20 g bactotryptone
• 5 g bacto-yeast extract
• 0.5 g NaCl
• 0.19 g KCl
• Adjust to pH 7.0 w/ NaOH

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)

LB Plates

LB Plates

(per/L H2O)

1. Weigh and add to H2O in large Erlenmeyer:

• 10 g Bacto-tryptone
• 5 g Bacto-yeast extract
• 10 g NaCl
• 15 g Bacto-agar

2. Autoclave on slow exhaust cycle 25 min

3. Cool in tap-water-bath 10 min, then place in 45ºC water bath. (in sterile hood)

4. When media has cooled to ~45ºC (comfortable to hold), add appropriate antibiotic.

5. Pour plates (~25 mL/plate)

6. Allow to solidify & store in bag in cold-room

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