Team:UIUC-Illinois/Notebook/Protocols/Bootcamp
From 2012.igem.org
(Difference between revisions)
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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/> | ||
- Add the following <br/> | - Add the following <br/> | ||
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- 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/><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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-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/> | ||
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<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/> | - We used the <a href="http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf">Ginkgo bioworks protocol</a> <br/><br/> | ||
- | - Control: 2 uL of Circular PSB1C3 plasmid, 13 uL ddH2O, 2 uL T4 ligase buffer, 1 uL T4 ligase | + | - Control: 2 uL of Circular PSB1C3 plasmid, 13 uL ddH2O, 2 uL T4 ligase buffer, 1 uL T4 ligase<br/> |
- | - P&C: 2 uL of Circular PSB1C3 plasmid, 2 uL of PUF, 11 uL ddH2O, 2 uL T4 ligase buffer, 1 uL T4 ligase | + | - P&C: 2 uL of Circular PSB1C3 plasmid, 2 uL of PUF, 11 uL ddH2O, 2 uL T4 ligase buffer, 1 uL T4 ligase<br/> |
- | - L&P: 2 uL of linear PSB1C3 plasmid, 2 uL of PUF, 11 uL ddH2O, 2 uL T4 ligase buffer, 1 uL T4 ligase | + | - L&P: 2 uL of linear PSB1C3 plasmid, 2 uL of PUF, 11 uL ddH2O, 2 uL T4 ligase buffer, 1 uL T4 ligase<br/> |
Revision as of 19:22, 1 June 2012