Team:IIT Madras/Notebook/Protocols/Miniprep using alkaline lysis buffers

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<h1 style="color:white;">IIT Madras</h1>
<h1 style="color:white;">IIT Madras</h1>
<h3 style="color:white;">And then the E.coli said, "Let there be light"</h3></div>
<h3 style="color:white;">And then the E.coli said, "Let there be light"</h3></div>
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<h1>Protocols</h1><br/>
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<h1>Miniprep using alkaline lysis buffers</h1><br/>
<p>For 5 ml Cultures: 12 – 16 hours incubation. <br/>
<p>For 5 ml Cultures: 12 – 16 hours incubation. <br/>
For 50 ml cultures: 8 hours of primary (2ml) and 16 hours of secondary (50 ml) add 50 ul.<br/>
For 50 ml cultures: 8 hours of primary (2ml) and 16 hours of secondary (50 ml) add 50 ul.<br/>
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<li>Discard isopropanol. Wash pellet with 70% ethanol. Spin and Drain Ethanol.</li>
<li>Discard isopropanol. Wash pellet with 70% ethanol. Spin and Drain Ethanol.</li>
<li>Let tubes dry at 37 C till the eppendorfs don’t smell of ethanol.</li>
<li>Let tubes dry at 37 C till the eppendorfs don’t smell of ethanol.</li>
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<li>Add 20 ul – 50 ul autoclaved MilliQ wateror (10mM Tris+1mM EDTA) with RNase. Leave for 30 mins at 43 C if RNase is added.</li>
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<li>Add 20 ul – 50 ul autoclaved MilliQ wateror (10mM Tris+1mM EDTA) with RNase. Leave for 30 mins at 43 C if RNase is added.</li></ul>
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<h3>Reagents</h3><br/>
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<p><b>Lysis Buffer 1</b></p>
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<ul>
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<li>50mM -- Glucose</li>
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<li>25mM -- TRIS-Cl (pH 8.0)</li>
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<li>10mM -- EDTA (pH 8.0)</li>
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</ul>
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<p>Autoclave and store at 4 C</p>
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<br/>
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<p><b>Lysis Buffer 2</b></p>
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<ul>
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<li>8ml -- Water</li>
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<li>1ml -- 10% SDS</li>
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<li>1ml -- 2N NaOH</li>
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</ul>
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<br/>
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<p><b>Lysis Buffer 3 (for 100ml)</b></p>
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<ul>
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<li>60ml -- 5M Sodium Acetate</li>
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<li>11.5ml -- Glacial Acetic Acid</li>
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<li>28.5ml -- Water</li>
</ul>
</ul>
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<p>Check pH. It should/will be around 5.2-5.4. Autoclave & store at 4 C.</p>
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<br/>
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Latest revision as of 12:56, 2 October 2011

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

Miniprep using alkaline lysis buffers


For 5 ml Cultures: 12 – 16 hours incubation.
For 50 ml cultures: 8 hours of primary (2ml) and 16 hours of secondary (50 ml) add 50 ul.

  • Pellet down the cells at 12000 rpm for 2 min. Use same eppendorf for 5 ml cultures. Remove all the media from pellet.
  • Add ice cold Soln 1, 250 ul, Vortex well. Incubate in ice for 5 min.
  • Add fresh soln II, 250 ul. Invert mix. Incubate at Room temperature for 5 min.
  • Add ice cold soln III, 250 ul. Invert mix. Incubate on ice for 5 min.
  • Add RNase, and incubate at 43 C for half an hour.
  • Add equal volume of Phenol Chloroform. Vortex well. Centrifuge at 12000 rpm for 10 minutes. Take the supernatant.
  • Add 0.6 volumes (~450 ul) of isopropanol. Shake well. Incubate at room temperature for 10 minutes. Centrifuge at 12000 rpm 4 C. 10 minutes.
  • Discard isopropanol. Wash pellet with 70% ethanol. Spin and Drain Ethanol.
  • Let tubes dry at 37 C till the eppendorfs don’t smell of ethanol.
  • Add 20 ul – 50 ul autoclaved MilliQ wateror (10mM Tris+1mM EDTA) with RNase. Leave for 30 mins at 43 C if RNase is added.

Reagents


Lysis Buffer 1

  • 50mM -- Glucose
  • 25mM -- TRIS-Cl (pH 8.0)
  • 10mM -- EDTA (pH 8.0)

Autoclave and store at 4 C


Lysis Buffer 2

  • 8ml -- Water
  • 1ml -- 10% SDS
  • 1ml -- 2N NaOH

Lysis Buffer 3 (for 100ml)

  • 60ml -- 5M Sodium Acetate
  • 11.5ml -- Glacial Acetic Acid
  • 28.5ml -- Water

Check pH. It should/will be around 5.2-5.4. Autoclave & store at 4 C.


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