Team:IIT Madras/Notebook/Protocols

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<body>
<div style="position:relative; top:50px;">
<div style="position:relative; top:50px;">
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<h1>Protocols</h1><br/>
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<h1><u>Protocols</u></h1><br/>
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<div id="protocol1">Preperation of competent DH5alpha cells</div>  
+
<div style="position:relative; left:16px;">
-
<div id="protocol2">Transformation</div>
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<div id="protocol1" style="cursor:pointer;"><b><u>Preparation of Competent DH5alpha Cells</u></b></div>
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<div id="protocol3">Miniprep using alkaline lysis buffers</div>
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<div id="protocol1_content" >
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<div id="protocol4">Agarose Gel Electrohoresis</div>
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<ul>
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<div id="protocol5">Restriction digestion</div>
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<li>Autoclave 0.1 M CaCl2 in 20% glycerol.</li>
-
<div id="protocol6">Gel Elusion</div>
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<li>Primary culture in 5 ml of LB broth for 16 hours.</li>
-
<div id="protocol7">PCR</div>
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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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<div id="protocol8">PCR_purification</div>
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<li>Incubate secondary cultures at 37 C in a shaker.</li>
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<div id="protocol9">Ligation</div>
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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>Re-suspend 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>Re-suspend 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>. Re-suspend 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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<br/>
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<div id="protocol2" style="cursor:pointer;"><b><u>Transformation</u></b></div>
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<div id="protocol2_content">
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<ul>
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<li>Add 2 ul of Plasmid DNA to competent cells (100 ul aliquot) on ice for 30 minutes.</li>
 +
<li>Heat shock : 90 s for 43 C.</li>
 +
<li>Keep on ice for 2 minutes.</li>
 +
<li>Add 900 ul of LB broth (sterile) into each microfuge tubes.</li>
 +
<li>Incubate in shaker at 37 C for 1 hour.</li>
 +
<li>Plate on plates (with antibiotics if necessary) and incubate at 37 C overnight.</li>
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</ul>
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</div>
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<br/>
 +
<div id="protocol3" style="cursor:pointer;"><b><u>Miniprep Using Alkaline Lysis Buffers</u></b></div>
 +
<div id="protocol3_content">
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<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/>
 +
<ul>
 +
<li>Pellet down the cells at 12000 rpm for 2 min. Use same eppendorf for 5 ml cultures. Remove all the media from pellet.</li>
 +
<li>Add ice cold Soln 1, 250 ul, Vortex well. Incubate in ice for 5 min.</li>
 +
<li>Add fresh soln II, 250 ul. Invert mix. Incubate at Room temperature for 5 min.</li>
 +
<li>Add ice cold soln III, 250 ul. Invert mix. Incubate on ice for 5 min.</li>
 +
<li>Add RNase, and incubate at 43 C for half an hour.</li>
 +
<li>Add equal volume of Phenol Chloroform. Vortex well. Centrifuge at 12000 rpm for 10 minutes. Take the supernatant.</li>
 +
<li>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.</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>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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<br/>
 +
<h3><b><u>Reagents</u></b></h3>
 +
 
 +
<p><b><u>Lysis Buffer 1</u></b></p>
 +
<ul>
 +
<li>50mM -- Glucose</li>
 +
<li>25mM -- TRIS-Cl (pH 8.0)</li>
 +
<li>10mM -- EDTA (pH 8.0)</li>
 +
</ul>
 +
<p>Autoclave and store at 4 C</p>
 +
<br/>
 +
 
 +
<p><b><u>Lysis Buffer 2</u></b></p>
 +
<ul>
 +
<li>8ml -- Water</li>
 +
<li>1ml -- 10% SDS</li>
 +
<li>1ml -- 2N NaOH</li>
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</ul>
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<br/>
 +
 
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<p><b><u>Lysis Buffer 3 (for 100ml)</u></b></p>
 +
<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>
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</ul>
 +
<p>Check pH. It should/will be around 5.2-5.4. Autoclave & store at 4 C.</p>
 +
<br/>
 +
</div>
 +
<div id="protocol6" style="cursor:pointer;"><b><u>Gel Elution</u></b></div>
 +
<div id="protocol6_content">
 +
<ul>
 +
<li>Prepare a 0.8% low melthing agarose gel</li>
 +
<li>Add 50 ul preparative reaction product into large wells</li>
 +
<li>Run the gel at 100 V for 30 – 45 mins</li>
 +
<li>Cut the gel with restricted DNA and keep it in an eppendorf</li>
 +
<li>To the cut DNA, add 3 volumes of Chaotropic salt. Incubate at 50 C for 10 minutes</li>
 +
<li>Add 10 ul of GPS (Glass Powder Solution) for a PCR product and 15 ul for a vector and
 +
incubate at Room Temperature for 10 mins. Shake the tubes continuously so that GPS is in suspension and doesn’t settle down.</li>
 +
<li>Centrifuge the tubes at 12000 rpm for 2 minutes and discard supernatant.</li>
 +
<li>Wash pellet by adding 50 volumes of wash buffer. That is, 500 ul of Wash buffer for insert
 +
and 750 ul for vector.</li>
 +
<li>Resuspend the pellet in 20 ul of water and heat at 50 C for 10 minutes. Centrifuge the tubes
 +
at 12000 rpm for 2 mins. Store supernatant at -20 C.</li></ul><br/><br/><br/><br/><br/>
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<li> Reference : Molecular Cloning, Sambrook and Russel </li>
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</div>
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Latest revision as of 00:30, 29 October 2011

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

Protocols


Preparation 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.
  • Re-suspend the pellet and keep on ice for 10 minutes.
  • Centrifuge at 6000 rpm for 10 min (4 C).
  • Re-suspend 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.
  • . Re-suspend 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
  • Add 2 ul of Plasmid DNA to competent cells (100 ul aliquot) on ice for 30 minutes.
  • Heat shock : 90 s for 43 C.
  • Keep on ice for 2 minutes.
  • Add 900 ul of LB broth (sterile) into each microfuge tubes.
  • Incubate in shaker at 37 C for 1 hour.
  • Plate on plates (with antibiotics if necessary) and incubate at 37 C overnight.

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.


Gel Elution
  • Prepare a 0.8% low melthing agarose gel
  • Add 50 ul preparative reaction product into large wells
  • Run the gel at 100 V for 30 – 45 mins
  • Cut the gel with restricted DNA and keep it in an eppendorf
  • To the cut DNA, add 3 volumes of Chaotropic salt. Incubate at 50 C for 10 minutes
  • Add 10 ul of GPS (Glass Powder Solution) for a PCR product and 15 ul for a vector and incubate at Room Temperature for 10 mins. Shake the tubes continuously so that GPS is in suspension and doesn’t settle down.
  • Centrifuge the tubes at 12000 rpm for 2 minutes and discard supernatant.
  • Wash pellet by adding 50 volumes of wash buffer. That is, 500 ul of Wash buffer for insert and 750 ul for vector.
  • Resuspend the pellet in 20 ul of water and heat at 50 C for 10 minutes. Centrifuge the tubes at 12000 rpm for 2 mins. Store supernatant at -20 C.





  • Reference : Molecular Cloning, Sambrook and Russel

    • Retrieved from "http://2011.igem.org/Team:IIT_Madras/Notebook/Protocols"