Team:UIUC-Illinois/Notebook/Protocols/Bootcamp
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<h2>Making LB for plates</h2> <br/> | <h2>Making LB for plates</h2> <br/> | ||
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. | + | 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/> | ||
- 5 g NaCl <br/> | - 5 g NaCl <br/> | ||
- | - 1.5 g Agar (NOT agarose!) <br/><br/> | + | - 1.5 g Agar (NOT agarose!)<br/><br/> |
- | 4. Then add 1 L of MilliQ water. | + | 4. Then add 1 L of MilliQ water. <br/> |
- | 5. Autoclave by total volume. | + | 5. Autoclave by total volume.<br/> |
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> <br/> | <h2>Making Liquid Media </h2> <br/> | ||
- | 1. Always use a 5-2.5-2.5 ratio of Tryptone to Yeast Extract to NaCl | + | 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 | + | 2. Add dry ingredients first, then add MilliQ water <br/> |
- | 3. No Agar! | + | 3. No Agar! <br/> |
4. Autoclave by total volume <br/><br/> | 4. Autoclave by total volume <br/><br/> | ||
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To make 400 mL of 10% glycerol you will need:<br/><br/> | To make 400 mL of 10% glycerol you will need:<br/><br/> | ||
- | -40 mL glycerol | + | -40 mL glycerol<br/> |
-360 mL of MilliQ water <br/><br/> | -360 mL of MilliQ water <br/><br/> | ||
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-320 mL of MilliQ water <br/><br/> | -320 mL of MilliQ water <br/><br/> | ||
- | 1. Use flasks and bottles before graduated cylinders (they take forever to mix!) | + | 1. Use flasks and bottles before graduated cylinders (they take forever to mix!)<br/> |
- | 2. Need a stir plate and a large stir bar | + | 2. Need a stir plate and a large stir bar <br/> |
- | 3. Stir until mixture is homogeneous | + | 3. Stir until mixture is homogeneous <br/> |
4. Don’t autoclave! <br/><br/> | 4. Don’t autoclave! <br/><br/> | ||
<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/> | ||
<h2>Autoclaving </h2><br/> | <h2>Autoclaving </h2><br/> | ||
- Robert is the source of official help on all things about the Autoclave. <br/><br/> | - Robert is the source of official help on all things about the Autoclave. <br/><br/> | ||
- | 1. Use the autoclave tape. | + | 1. Use the autoclave tape. <br/> |
- | 2. Look at the Betastar for settings. | + | 2. Look at the Betastar for settings. <br/> |
- | 3. Go by materials and total volume. | + | 3. Go by materials and total volume. <br/> |
4. Make sure the autoclave tape has changed colour by the end of the cycle. Check conditions and settings then repeat steps 1-4 if tape has not changed colour. <br/><br/> | 4. Make sure the autoclave tape has changed colour by the end of the cycle. Check conditions and settings then repeat steps 1-4 if tape has not changed colour. <br/><br/> | ||
<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