Team:Amsterdam/Labwork/Protocols

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==P1. Making LB Medium==
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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.
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===Ingredients===
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#10 g Bacto-tryptone
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#5 g yeast extract
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#10 g NaCl
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''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.
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===Protocol===
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#Mix dry ingredients and add distilled water up to 1 Liter
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#Pour into 2 L flask (or greater)
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#Autoclave (liquid cycle)
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#* 250°'''F''', 22psi, 30 minutes
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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.
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===Source===
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Adapted From:
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J. Sambrook, D.W. Russell, ''Molecular Cloning: A Laboratory Manual'' (Cold Spring Harbor Laboratory Press, New York, ed. 3, 2001) pg. A2.2
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Revision as of 13:42, 20 June 2011

Contents

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

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