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| | {{:Team:Amsterdam/Header}} | | {{:Team:Amsterdam/Header}} |
| - | ==P1. Making LB Medium== | + | =Protocols= |
| - | 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.
| + | ===Preparations=== |
| | + | *[[Team:Amsterdam/Notebook/Protocols/Preparing_Media|Preparing media]] |
| | + | *[[Team:Amsterdam/Notebook/Protocols/Making_Competent_Cells|Preparation of chemically competent cells]] |
| | + | *[[Team:Amsterdam/Notebook/Protocols/Making_electrocompetent_Cells|Preparation of electrocompetent cells]] |
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| - | ===Ingredients=== | + | ===Cloning=== |
| | + | *[[Team:Amsterdam/Notebook/Protocols/Miniprepping|Miniprepping]] |
| | + | *[[Team:Amsterdam/Notebook/Protocols/Digestion|Digestion]] |
| | + | *[[Team:Amsterdam/Notebook/Protocols/Ligations|Ligations]] |
| | + | *[[Team:Amsterdam/Notebook/Protocols/Transforming_Competent_Cells|Transforming chemically competent cells]] |
| | + | *[[Team:Amsterdam/Notebook/Protocols/Transforming_electrocompetent_Cells|Transforming electrocompetent cells]] |
| | + | *[[Team:Amsterdam/Notebook/Protocols/ColonyPCR|Colony PCR]] |
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| - | #10 g Bacto-tryptone
| + | ===BioBrick characterization=== |
| - | #5 g yeast extract
| + | *[[Team:Amsterdam/Notebook/Protocols/Growth_Curve|Growth curve measurement]] |
| - | #10 g NaCl
| + | *[[Team:Amsterdam/Notebook/Protocols/Freeze_thaw|Freeze/thaw survival measurement]] |
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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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| - | ==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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| - |
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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
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| - | ==P3. Making SOC Medium==
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| - | SOC Medium.
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| - | ===Ingredients===
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| - | *[[Team:Amsterdam/Notebook/Protocols#P2. Making SOB Medium|P2. Making SOB Medium]]
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| - | *20 mM glucose
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| - | ===Protocol===
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| - | #Follow directions to make 1 liter of [[Team:Amsterdam/Notebook/Protocols#P2. Making SOB Medium|P2. Making SOB Medium]] media
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| - | #After cooling medium to less than 50°C, add 20 ml filter sterilized 20% glucose solution
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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
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| | {{:Team:Amsterdam/Footer}} | | {{:Team:Amsterdam/Footer}} |
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