Difference between revisions of "Team:Glasgow/Protocols"

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<li>Cells were kept on ice for 2 minutes</li>
 
<li>Cells were kept on ice for 2 minutes</li>
 
<li>200μl of broth was added and the cells incubated at 37⁰C for expression step (time varies dependent on antibiotic resistance gene in the plasmid that has just been transformed into the cells):
 
<li>200μl of broth was added and the cells incubated at 37⁰C for expression step (time varies dependent on antibiotic resistance gene in the plasmid that has just been transformed into the cells):
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<ul>
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<table>
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<li>Chloramphenicol resistance = 90 minutes</li>
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<tr>
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<li>Kanamycin resistance = 60 minutes</li>
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<th>Resistance</th>
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<li>Ampicillin resistance = 30 minutes</li>
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<th>Time</th>
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</ul>
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</tr>
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<tr>
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<td>Chloramphenicol</td>
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<td>90 minutes</td>
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</tr>
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<tr>
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<td>Kanamycin</td>
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<td>60 minutes</td>
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</tr>
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<tr>
 +
<td>Ampicillin</td>
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<td>30 minutes</td>
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</tr>
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</table>
 
</li>
 
</li>
 
<li>100-200μl of transformed cells was spread on dried L-agar plates with required antibiotic(s) to select for plasmid(s) </li>
 
<li>100-200μl of transformed cells was spread on dried L-agar plates with required antibiotic(s) to select for plasmid(s) </li>
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<li>Incubate at 37⁰C, shaking at 250rpm until mid-log phase (OD600=0.5-0.7)</li>
 
<li>Incubate at 37⁰C, shaking at 250rpm until mid-log phase (OD600=0.5-0.7)</li>
 
<li>Split culture into 2 x 200ml samples</li>
 
<li>Split culture into 2 x 200ml samples</li>
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<li>Chill on ice for 20mins</li>
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<li>Chill on ice for 20 minutes</li>
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<li>Spin 4000g for 15minutes at 4⁰C</li>
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<li>Spin 4000g for 15 minutes at 4⁰C</li>
 
<li>Re-suspend each in 200ml ice cold 10% glycerol</li>
 
<li>Re-suspend each in 200ml ice cold 10% glycerol</li>
−
<li>Spin 4000g for 15minutes at 4⁰C</li>
+
<li>Spin 4000g for 15 minutes at 4⁰C</li>
 
<li>Re-suspend each in 100ml ice cold 10% glycerol</li>
 
<li>Re-suspend each in 100ml ice cold 10% glycerol</li>
−
<li>Spin 4000g for 15minutes at 4⁰C</li>
+
<li>Spin 4000g for 15 minutes at 4⁰C</li>
 
<li>Re-suspend each in 10ml ice cold 10% glycerol</li>
 
<li>Re-suspend each in 10ml ice cold 10% glycerol</li>
−
<li>Spin 4000g for 15minutes at 4⁰C</li>
+
<li>Spin 4000g for 15 minutes at 4⁰C</li>
 
<li>Re-suspend each in 500μl ice cold 10% glycerol</li>
 
<li>Re-suspend each in 500μl ice cold 10% glycerol</li>
 
<li>Aliquot 60μl into small eppendorf tubes and store at -70⁰C</li>
 
<li>Aliquot 60μl into small eppendorf tubes and store at -70⁰C</li>
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<li>Bake in the oven for 15-20 minutes, or until pale golden-brown. Set aside to cool on a wire rack.</li>
 
<li>Bake in the oven for 15-20 minutes, or until pale golden-brown. Set aside to cool on a wire rack.</li>
 
</ol>
 
</ol>
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</div>
 
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</div>
 
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<div class="accordion-item" data-accordion-item>
 
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<a href="#" class="accordion-title">Protocol Two</a>
 
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<div class="accordion-content" data-tab-content>
 
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Step One<br>
 
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Step Two<br>
 
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etc
 
 
</div>
 
</div>
 
</div>
 
</div>

Revision as of 14:22, 1 August 2016

Glasgow iGEM 2016
Protocols

Here's the techniques we used blah blah blah"

Preparation of CaCl2 Competent Cells
  1. Dilute 400μl of overnight liquid culture into 20ml of broth with any necessary antibiotics to select for any plasmids already transformed into the cells
  2. Incubate at 37⁰C, shaking at 225rpm for 90 minutes
  3. Spin down for 2 minutes at 7000rpm at 4⁰C
  4. Discard supernatant, resuspend pellet in 10ml of 50 mM CaCl2, keep on ice
  5. Repeat centrifugation for 2 minutes at 7000rpm at 4⁰C
  6. Discard supernatant and the resuspend pellet in 1ml of 50 mM CaCl2, keep on ice
  7. CaCl2 competent cells can be kept on ice in the fridge overnight
Transformation of CaCl2 Competent Cells
  1. 1μl of plasmid DNA was added to 100μl of competent cells
  2. Samples were incubated on ice for 20 minutes
  3. Heat shock was cried out at 37⁰C for 5 minutes
  4. Cells were kept on ice for 2 minutes
  5. 200μl of broth was added and the cells incubated at 37⁰C for expression step (time varies dependent on antibiotic resistance gene in the plasmid that has just been transformed into the cells):
    Resistance Time
    Chloramphenicol 90 minutes
    Kanamycin 60 minutes
    Ampicillin 30 minutes
  6. 100-200μl of transformed cells was spread on dried L-agar plates with required antibiotic(s) to select for plasmid(s)
  7. Plates were incubated at 37°C overnight
Preparation of Electrocompetent Cells
  1. Inoculate 400ml L-broth with 4ml culture
  2. Incubate at 37⁰C, shaking at 250rpm until mid-log phase (OD600=0.5-0.7)
  3. Split culture into 2 x 200ml samples
  4. Chill on ice for 20 minutes
  5. Spin 4000g for 15 minutes at 4⁰C
  6. Re-suspend each in 200ml ice cold 10% glycerol
  7. Spin 4000g for 15 minutes at 4⁰C
  8. Re-suspend each in 100ml ice cold 10% glycerol
  9. Spin 4000g for 15 minutes at 4⁰C
  10. Re-suspend each in 10ml ice cold 10% glycerol
  11. Spin 4000g for 15 minutes at 4⁰C
  12. Re-suspend each in 500μl ice cold 10% glycerol
  13. Aliquot 60μl into small eppendorf tubes and store at -70⁰C
Shortbread
A good supply of shortbread is essential to any productive lab environment.
  1. Heat the oven to 190C/375F/Gas 5
  2. Beat the butter and the sugar together until smooth.
  3. Stir in the flour to get a smooth paste. Turn on to a work surface and gently roll out until the paste is 1cm/½in thick.
  4. Cut into rounds or fingers and place onto a baking tray. Sprinkle with caster sugar and chill in the fridge for 20 minutes.
  5. Bake in the oven for 15-20 minutes, or until pale golden-brown. Set aside to cool on a wire rack.