Team:Amsterdam/Labwork/Protocols

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Contents 1 P1. Making LB Medium 1.1 Ingredients 1.2 Protocol 1.3 Source 2 P2. Making SOB Medium 2.1 Ingredients 2.2 15/10 medium 2.3 Source 3 P3. Making SOC Medium 4 Ingredients 5 Protocol 6 Source

P1. 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

P2. 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

P3. Making SOC Medium

SOC Medium.

Ingredients

• P2. Making SOB Medium
• 20 mM glucose

Protocol

1. Follow directions to make 1 liter of P2. Making SOB Medium media
2. After cooling medium to less than 50°C, add 20 ml filter sterilized 20% glucose solution

Source

Adapted From:

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