Team:Amsterdam/Notebook/Protocols/Preparing Media

From 2011.igem.org

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#5 g yeast extract
#5 g yeast extract
#10 g NaCl
#10 g NaCl
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<br>
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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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''Note: To make agar plates add 1,5 gram bacto-agar to 1 liter LB medium.''
===Protocol===
===Protocol===
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#Autoclave (liquid cycle)
#Autoclave (liquid cycle)
#* 250&deg;'''F''', 22psi, 30 minutes
#* 250&deg;'''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.
 
===Source===
===Source===
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==Making SOB Medium==
==Making SOB Medium==
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SOB Medium.  Used in growing bacteria for preparing chemically competent cells
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SOB Medium.  Used in growing bacteria for preparing chemically competent cells.
=== Ingredients ===
=== Ingredients ===
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*2.4 g MgSO<sub>4</sub>
*2.4 g MgSO<sub>4</sub>
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''Note:'' Some formulations of SOB use 10 mM MgCl<sub>2</sub> and 10 mM MgSO<sub>4</sub> instead of 20 mM MgSO<sub>4</sub>.
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''Note:Adjust to pH 7.5 prior to use.  This requires approximately 25 ml of 1M NaOH per liter.''
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SOB medium is also available dry premixed from Difco, 0443-17.
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Adjust to pH 7.5 prior to use.  This requires approximately 25 ml of 1M NaOH per liter.
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===15/10 medium===
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Growth of competent TOP10 cells in Example 2 of the Bloom05 patent is performed in 15/10 broth, which is similar to SOB:
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* 1.5% yeast extract
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* 1% Bacto-Tryptone
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* 10mM NaCl
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* 2mM KCl
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* 10 mM MgCl<sub>2</sub>
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* 10 mM MgSO<sub>4</sub>
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===Source===
===Source===
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===Ingredients===
===Ingredients===
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*[[Team:Amsterdam/Notebook/Protocols#Making SOB Medium (P2.)|SOB]]
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*SOB
*20 mM glucose
*20 mM glucose
===Protocol===
===Protocol===
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#Follow directions to make 1 liter of [[Team:Amsterdam/Notebook/Protocols#Making SOB Medium (P2.)|Making SOB Medium (P2.)]] media.
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#Follow directions to make 1 liter of SOB medium.
#After cooling medium to less than 50&deg;C, add 20 ml filter sterilized 1M glucose solution.
#After cooling medium to less than 50&deg;C, add 20 ml filter sterilized 1M glucose solution.

Revision as of 10:04, 19 September 2011

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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