Team:UIUC-Illinois/Notebook/Protocols/Bootcamp
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To make 1 Liter of LB: <br/><br/> | To make 1 Liter of LB: <br/><br/> | ||
1. Always use a 10:5:5 ratio of Tryptone to Yeast Extract to NaCl. <br/> | 1. Always use a 10:5:5 ratio of Tryptone to Yeast Extract to NaCl. <br/> | ||
- | 2. Add dry ingredients first. | + | 2. Add dry ingredients first. <br/> |
- | 3. Use a 2L flask. <br/> | + | 3. Use a 2L flask. <br/> <br/> |
- | + | 4. Add the following <br/><br/> | |
- 10 g Tryptone <br/> | - 10 g Tryptone <br/> | ||
- 5 g Yeast Extract <br/> | - 5 g Yeast Extract <br/> | ||
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6. Pour 25 mL on each plate (just enough to cover the bottom). <br/><br/> | 6. Pour 25 mL on each plate (just enough to cover the bottom). <br/><br/> | ||
- | <h2>Making Liquid Media </h2 | + | <h2>Making Liquid Media </h2> <br/> |
1. Always use a 5-2.5-2.5 ratio of Tryptone to Yeast Extract to NaCl <br/> | 1. Always use a 5-2.5-2.5 ratio of Tryptone to Yeast Extract to NaCl <br/> | ||
2. Add dry ingredients first, then add MilliQ water <br/> | 2. Add dry ingredients first, then add MilliQ water <br/> | ||
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<h2>Making Glycerol Stock </h2> <br/> | <h2>Making Glycerol Stock </h2> <br/> | ||
- | To make 400 mL of 10% glycerol you will need:<br/> | + | To make 400 mL of 10% glycerol you will need:<br/><br/> |
-40 mL glycerol<br/> | -40 mL glycerol<br/> | ||
-360 mL of MilliQ water <br/><br/> | -360 mL of MilliQ water <br/><br/> | ||
- | To make 400 mL of 20% glycerol you will need:<br/> | + | To make 400 mL of 20% glycerol you will need:<br/><br/> |
-80 mL glycerol<br/> | -80 mL glycerol<br/> | ||
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<h2>Making ddH2O</h2> <br/> | <h2>Making ddH2O</h2> <br/> | ||
- | - Use the small bottles. | + | - Use the small bottles. <br/> |
- Autoclave water by total volume. <br/><br/> | - Autoclave water by total volume. <br/><br/> | ||
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<h2>Ligation </h2><br/> | <h2>Ligation </h2><br/> | ||
- | + | We used the <a href="http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf">Ginkgo bioworks protocol</a> <br/><br/> | |
- | + | 1. Control: <br/><br/> | |
- | + | - 2 uL of Circular PSB1C3 plasmid 13 uL ddH2O <br/> | |
- | + | - 2 uL T4 ligase buffer<br/> | |
+ | - 1 uL T4 ligase <br/><br/> | ||
+ | 2. P&C: <br/><br/> | ||
+ | - 2 uL of Circular PSB1C3 plasmid<br/> | ||
+ | - 2 uL of PUF, 11 uL ddH2O<br/> | ||
+ | - 2 uL T4 ligase buffer<br/> | ||
+ | - 1 uL T4 ligase<br/><br/> | ||
+ | 3. L&P:<br/><br/> | ||
+ | - 2 uL of linear PSB1C3 plasmid<br/> | ||
+ | - 2 uL of PUF<br/> | ||
+ | - 11 uL ddH2O<br/> | ||
+ | - 2 uL T4 ligase buffer<br/> | ||
+ | - 1 uL T4 ligase<br/><br/> | ||
Latest revision as of 15:31, 4 June 2012