Team:NGSS TR/isolation.html

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<h1>PROTOCOLS</h1>
 
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<h1>PROTOCOLS</h1>
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     <h2><span class="gfdg">4.ISOLATION</span></h2>
     <h2><span class="gfdg">4.ISOLATION</span></h2>

Latest revision as of 11:34, 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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