Team:Amsterdam/Notebook/Protocols/Preparing Media

From 2011.igem.org

Contents

Making LB Medium

Luria-Bertani Medium (aka L-Broth or LB Medium). (Bertani says LB really stands for lysogeny broth.) LB is a standard growth medium for a variety of bacteria and conditions.

Ingredients

  1. 10 g Bacto-tryptone
  2. 5 g yeast extract
  3. 10 g NaCl


Note: To make agar plates add 1,5 gram bacto-agar to 1 liter LB medium.

Protocol

  1. Mix dry ingredients and add distilled water up to 1 Liter
  2. Pour into 2 L flask (or greater)
  3. Autoclave (liquid cycle)
    • 250°F, 22psi, 30 minutes

Source

Adapted From:

J. Sambrook, D.W. Russell, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, ed. 3, 2001) pg. A2.2

Making SOB Medium

SOB Medium. Used in growing bacteria for preparing chemically competent cells.

Ingredients

  • 0.5% (w/v) yeast extract
  • 2% (w/v) tryptone
  • 10 mM NaCl
  • 2.5 mM KCl
  • 20 mM MgSO4

Per liter:

  • 5 g yeast extract
  • 20 g tryptone
  • 0.584 g NaCl
  • 0.186 g KCl
  • 2.4 g MgSO4

Note:Adjust to pH 7.5 prior to use. This requires approximately 25 ml of 1M NaOH per liter.

Source

Adapted From:

F. Ausubel et al., Short Protocols in Molecular Biology (John Wiley & Sons, ed. 4, 1999) pg. A1-36

Making SOC Medium

SOC Medium used during transformations.

Ingredients

  • SOB
  • 20 mM glucose

Protocol

  1. Follow directions to make 1 liter of SOB medium.
  2. After cooling medium to less than 50°C, add 20 ml filter sterilized 1M glucose solution.

Source

Adapted From:

F. Ausubel et al., Short Protocols in Molecular Biology (John Wiley & Sons, ed. 4, 1999) pg. A1-36

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