Team:Amsterdam/Labwork/Protocols

From 2011.igem.org

(Difference between revisions)
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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
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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==P2. Making SOB Medium==
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SOB Medium.  Used in growing bacteria for preparing chemically competent cells
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== Ingredients ==
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*0.5% (w/v) yeast extract
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*2% (w/v) tryptone
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*10 mM NaCl
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*2.5 mM KCl
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*20 mM MgSO<sub>4</sub>
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Per liter:
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*5 g yeast extract
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*20 g tryptone
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*0.584 g NaCl
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*0.186 g KCl
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*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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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==
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Adapted From:
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F. Ausubel et al., ''Short Protocols in Molecular Biology'' (John Wiley & Sons, ed. 4, 1999) pg. A1-36
{{:Team:Amsterdam/Footer}}
{{:Team:Amsterdam/Footer}}

Revision as of 13:45, 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

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

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