Team:IIT Madras/Notebook/Protocols

From 2011.igem.org

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<div id="protocol1">Preperation of competent DH5alpha cells</div>
<div id="protocol1">Preperation of competent DH5alpha cells</div>
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<div id="protocol1_content"></div>
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<div id="protocol1_content">
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<ul>
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<li>Autoclave 0.1 M CaCl2 in 20% glycerol.</li>
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<li>Primary culture in 5 ml of LB broth for 16 hours.</li>
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<li>Add 1 ml of primary culture into 50 ml of media for secondary culture.<br/>Check OD at 600 nm.</li>
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<li>Incubate secondary cultures at 37 C in a shaker.</li>
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<li>Check OD600 after 1 hour, 1.5 hours, 2 hours.</li>
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<li>Keep the cells on ice (for 10 minutes) for harvesting as soon as OD600 reaches 0.4 (0.4 -0.6 is the range).</li>
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<li>Pellet down the cells at 6000 rpm for 10 min (4 C ). <br/>(Each 50 ml culture can be added to 50 ml falcon tubes)</li>
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<li>Add 10 ml of ice cold 0.1M CaCl2.</li>
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<li>Resuspend the pellet and keep on ice for 10 minutes.</li>
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<li>Centrifuge at 6000 rpm for 10 min (4 C).</li>
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<li>Resuspend the pellet in 5 ml ice cold 0.1 M CaCl2 Solution.</li>
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<li>Again, keep on ice for 10 min. and centrifuge at 6000 rpm for 10 min.</li>
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<li>. Resuspend the pellet in 2 ml ice cold 0.1 M CaCl2 Solution. Alliquot 100 ul into a number of tubes and store them at -80 C.</li>
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</ul>
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</div>
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<div id="protocol2">Transformation</div>
<div id="protocol2">Transformation</div>
<div id="protocol2_content"></div>  
<div id="protocol2_content"></div>  
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<div id="protocol3">Miniprep using alkaline lysis buffers</div>
<div id="protocol3">Miniprep using alkaline lysis buffers</div>
<div id="protocol3_content"></div>
<div id="protocol3_content"></div>
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<div id="protocol4">Agarose Gel Electrohoresis</div>
<div id="protocol4">Agarose Gel Electrohoresis</div>
<div id="protocol4_content"></div>
<div id="protocol4_content"></div>
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<div id="protocol5">Restriction digestion</div>
<div id="protocol5">Restriction digestion</div>
<div id="protocol5_content"></div>
<div id="protocol5_content"></div>
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<div id="protocol6">Gel Elusion</div>
<div id="protocol6">Gel Elusion</div>
<div id="protocol6_content"></div>
<div id="protocol6_content"></div>
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<div id="protocol7">PCR</div>
<div id="protocol7">PCR</div>
<div id="protocol7_content"></div>
<div id="protocol7_content"></div>
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<div id="protocol8">PCR_purification</div>
<div id="protocol8">PCR_purification</div>
<div id="protocol8_content"></div>
<div id="protocol8_content"></div>
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<div id="protocol9">Ligation</div>
<div id="protocol9">Ligation</div>
<div id="protocol9_content"></div>
<div id="protocol9_content"></div>
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</div>
</div>
</html>
</html>

Revision as of 20:22, 5 October 2011

bar iGEM 2011 - Home Page Indian Institute of Technology - Madras

Protocols


Preperation of competent DH5alpha cells
  • Autoclave 0.1 M CaCl2 in 20% glycerol.
  • Primary culture in 5 ml of LB broth for 16 hours.
  • Add 1 ml of primary culture into 50 ml of media for secondary culture.
    Check OD at 600 nm.
  • Incubate secondary cultures at 37 C in a shaker.
  • Check OD600 after 1 hour, 1.5 hours, 2 hours.
  • Keep the cells on ice (for 10 minutes) for harvesting as soon as OD600 reaches 0.4 (0.4 -0.6 is the range).
  • Pellet down the cells at 6000 rpm for 10 min (4 C ).
    (Each 50 ml culture can be added to 50 ml falcon tubes)
  • Add 10 ml of ice cold 0.1M CaCl2.
  • Resuspend the pellet and keep on ice for 10 minutes.
  • Centrifuge at 6000 rpm for 10 min (4 C).
  • Resuspend the pellet in 5 ml ice cold 0.1 M CaCl2 Solution.
  • Again, keep on ice for 10 min. and centrifuge at 6000 rpm for 10 min.
  • . Resuspend the pellet in 2 ml ice cold 0.1 M CaCl2 Solution. Alliquot 100 ul into a number of tubes and store them at -80 C.
Transformation
Miniprep using alkaline lysis buffers
Agarose Gel Electrohoresis
Restriction digestion
Gel Elusion
PCR
PCR_purification
Ligation

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