Team:Amsterdam/Notebook/Protocols/Preparing Media

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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: There are two formulations of LB, Miller and Lennox, that differ in the amount of NaCl. Lennox has less salt, only 5 g/L. The Qiagen miniprep kit recommends LB with 10 g NaCl for highest plasmid yields.

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

Notes: We do not pH medium when we make it on the fly. However, if it is really important, pH the medium to 7.0 with 5M NaOH (~200µL). We usually obtain this from the kitchen.

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: Some formulations of SOB use 10 mM MgCl2 and 10 mM MgSO4 instead of 20 mM MgSO4.

SOB medium is also available dry premixed from Difco, 0443-17.

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

15/10 medium

Growth of competent TOP10 cells in Example 2 of the Bloom05 patent is performed in 15/10 broth, which is similar to SOB:

  • 1.5% yeast extract
  • 1% Bacto-Tryptone
  • 10mM NaCl
  • 2mM KCl
  • 10 mM MgCl2
  • 10 mM MgSO4

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 Making SOB Medium (P2.) media.
  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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