Team:NGSS TR/isolation.html

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<h1>PROTOCOLS</h1>
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  <blockquote>
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    <h2><span class="gfdg">4.ISOLATION</span></h2>
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    <blockquote>
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      <p>1-Liquid cultures centrifuge at 13000 rpm 10 minuites.<br />
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        2-  After the  centrifuge decant the supernatant and remove all medium residue by pipet.<br />
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        3-Completely resuspend the cell pellet in 250 ul of  resuspension solution by voretxing. <br />
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        4-Add 250 ul lysis solution and invert 4-6 times to lyse the  cells. Incubate at room temperature for 3 minutes.<br />
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        5-Add 350 ul neutralization solution and invert 4-6 times.<br />
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        6- Centrifuge at 13000 rpm, 5 minutes.<br />
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        7-  Transfer the  supernatant carrefully to the coloumn. <br />
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        8- Centrifuge at 13000 rpm, 1 minute. Discard the flow  through. <br />
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        9-  Wash the coloumn  once with 500 ul wash solution by centrifuging   for 60 seconds at 13000 rpm and discard the flow through.<br />
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        10- Repeat  the wash  procedure (step 9)<br />
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        11-Transfer the coloumn into a fresh 1.5 ml centrifuge tube <br />
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        12- Add 50 ul elution buffer <br />
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        13- Incubate for 2 min at room temperature and centrifuge  for 2 min (13000 rpm) </p>
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    </blockquote>
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  <p>
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    <blockquote>
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</blockquote>

Revision as of 11:33, 20 June 2014

PROTOCOLS

4.ISOLATION

1-Liquid cultures centrifuge at 13000 rpm 10 minuites.
2-  After the centrifuge decant the supernatant and remove all medium residue by pipet.
3-Completely resuspend the cell pellet in 250 ul of resuspension solution by voretxing.
4-Add 250 ul lysis solution and invert 4-6 times to lyse the cells. Incubate at room temperature for 3 minutes.
5-Add 350 ul neutralization solution and invert 4-6 times.
6- Centrifuge at 13000 rpm, 5 minutes.
7-  Transfer the supernatant carrefully to the coloumn.
8- Centrifuge at 13000 rpm, 1 minute. Discard the flow through.
9-  Wash the coloumn once with 500 ul wash solution by centrifuging  for 60 seconds at 13000 rpm and discard the flow through.
10- Repeat  the wash procedure (step 9)
11-Transfer the coloumn into a fresh 1.5 ml centrifuge tube
12- Add 50 ul elution buffer
13- Incubate for 2 min at room temperature and centrifuge for 2 min (13000 rpm)

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