Difference between revisions of "Team:UMaryland/Notebook2"

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<!--Attention! If you are not part of the website team, you are NOT allowed to touch anything above this line without the express permission of Best Kohai.-->
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<p style="font-size:64px"><b>Protocols</b>
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<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
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<b>Miniprep</b></a>
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<p style="font-size:24px;text-align:left;text-decoration: underline;">
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Materials:
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<ul class="a">
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  <li>- 250 µL Buffer P1</li>
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  <li>- 250 µLBuffer P2</li>
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  <li>- 350 µL Buffer N3</li>
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  <li>- 750 µL Buffer PE</li>
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  <li>- 100 µL DDH2O</li>
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</ul>
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<p style="font-size:24px;text-align:left;text-decoration: underline;">
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Procedure:
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<ul class="a">
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  <li>- Pellet 1 mL bacterial overnight culture 5 times ( using a 1.5 mL centrifuge tube) by centrifugation at max speed (13000 rpm) for 60 secs room temperature (15 - 25)</li>
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  <li>- Resuspend pellet in 250 µL Buffer P1</li>
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  <li>- Add 250 µLBuffer P2 and mix thoroughly by inverting the tube 4-6 times until solution becomes clear </li>
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  <li>- Do not allow reaction to proceed for more than 5 mins</li>
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  <li>- If using Lyse Blue, reagent, solution will turn blue</li>
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  <li>- Add 350 µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times
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  </li>
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  <li>- Centrifuge for 10 mins at 13000 rpm</li>
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  <li>- Apply 800 µLsupernatant from step 5 to Q1A prep 2.0 spin column by pipetting</li>
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  <li>- Centrifuge for 60 secs and discard flow through</li>
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  <li>- Wash the Q1A prep column with 750 µL Buffer PE</li>
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  <li>- Centrifuge for 60 secs and discard flow through</li>
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  <li>- Centrifuge for 60 secs to remove residual wash buffer</li>
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  <li>- To elute DNA, add 50 µL DDH2O to the center of Q1A prep column</li>
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  <li>- Let stand for 1 min and centrifuge for 1 min</li>
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  <li>- Repeat steps 12 and 13</li>
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</ul>
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<br>
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<br>
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</div>
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Revision as of 10:05, 18 September 2015