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Team:Amsterdam/Labwork/Protocols
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===Source=== | ===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 == | ||
| + | *[[Team:Amsterdam/Notebook/Protocols#P2. Making SOB Medium|P2. Making SOB Medium]] | ||
| + | *20 mM glucose | ||
| + | |||
| + | ==Protocol== | ||
| + | #Follow directions to make 1 liter of [[Team:Amsterdam/Notebook/Protocols#P2. Making SOB Medium|P2. Making SOB Medium]] media | ||
| + | #After cooling medium to less than 50°C, add 20 ml filter sterilized 20% glucose solution | ||
| + | |||
| + | ==Source== | ||
Adapted From: | Adapted From: | ||
Revision as of 13:53, 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
- 10 g Bacto-tryptone
- 5 g yeast extract
- 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
- Mix dry ingredients and add distilled water up to 1 Liter
- Pour into 2 L flask (or greater)
- 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
- Follow directions to make 1 liter of P2. Making SOB Medium media
- 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
