Team:NGSS TR/digestion.html

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<h1>PROTOCOLS</h1>
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<blockquote>
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  <h2><span class="gfdg">1.DIGESTION PROTOCOL</span></h2>
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  <blockquote>
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    <h2> -Procedure </h2>
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  </blockquote>
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  <p>&nbsp;</p>
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  <ol>
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    <li>Keep all enzymes and buffers used on ice.</li>
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    <li>Add 500ng of DNA to the appropriately labelled tube. Add  distilled water to the tubes for a total volume of 40ul in each tube.</li>
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  </ol>
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  <div>
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    <p>Pipet 5ul of NEB Buffer 2 to each tube. Calculation example (with 50ng/ul as DNA sample concentration):</p>
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    <p>500ng ÷ 50ng/ul = 10ul of DNA sample</p>
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    <p>50ul (total volume) – 10ul (DNA sample) = 40ul of distilled water</p>
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  </div>
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  <ol>
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    <li>Pipet 5ul of NEB Buffer 2 to each tube.</li>
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    <li>Pipet 0.5ul of BSA to each tube.</li>
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    <li>Add  1ul enzyme 1</li>
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    <li>Add 1ul enzyme 2</li>
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    <li>The total volume in each tube should be approximately 50ul. Mix  well by pipetting slowly up and down.</li>
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    <li>Incubate the restriction digests at 37°C for 35 minutes, then 80°C for 20 minutes.</li>
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  </ol>
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  <p>                                                          </p>
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  <p>&nbsp;</p>
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<p>&nbsp;</p>
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</blockquote>

Latest revision as of 11:13, 20 June 2014

PROTOCOLS

1.DIGESTION PROTOCOL

-Procedure

 

  1. Keep all enzymes and buffers used on ice.
  2. Add 500ng of DNA to the appropriately labelled tube. Add distilled water to the tubes for a total volume of 40ul in each tube.

Pipet 5ul of NEB Buffer 2 to each tube. Calculation example (with 50ng/ul as DNA sample concentration):

500ng ÷ 50ng/ul = 10ul of DNA sample

50ul (total volume) – 10ul (DNA sample) = 40ul of distilled water

  1. Pipet 5ul of NEB Buffer 2 to each tube.
  2. Pipet 0.5ul of BSA to each tube.
  3. Add  1ul enzyme 1
  4. Add 1ul enzyme 2
  5. The total volume in each tube should be approximately 50ul. Mix well by pipetting slowly up and down.
  6. Incubate the restriction digests at 37°C for 35 minutes, then 80°C for 20 minutes.

                                                         

 

 

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