Difference between revisions of "Team:UMaryland/protocols"

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   <li>- 37° for 30 minutes</li>
 
   <li>- 37° for 30 minutes</li>
 
   <li>- 80° for 20 minutes</li>   
 
   <li>- 80° for 20 minutes</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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<div id='contentbox'>
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<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
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<b>Gel Extract</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>- </li>
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  <li>- </li>
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  <li>- </li>
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  <li>- </li>
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  <li>- </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>- cut out gel portions with scalpel</li>
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  <li>- weigh gel slice in colorless tube. Add 3 volumes of QG buffer to 1 volume of gel</li>
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  <li>- Incubate at 50 for 10 mins until gels slice dissolves. Mix by vortexing tube every 2-3 mins during incubation to help the gel dissolve
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</li>
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  <li>- After the gel dissolves completely, check color of the mixture is yellow (it may be orange or violet)
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</li>
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  <li>- Add 10 µL of 3M sodium acetate (ph 5.0) to make solution more yellow (regardless of current solution color)</li>
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  <li>- The color of solution relates to ph indicator in Q3</li>
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  <li>- Add 1 gel volume of of isopropyl to the sample and mix. This increases yield of DNA fragments between 500 bp and 4KB</li>
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  <li>- Place a Q1Aquick spin column in a provided 2ml collection tube</li>
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  <li>- Apply sample to spin column, centrifuge for 1 min to bind dna</li>
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  <li>- discard flow through</li>
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  <li>- wash with .75 mL buffer PE in column, centrifuge 1 minute</li>
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  <li>- Place column in a clean 1.5 microcentrifuge tube</li>
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  <li>- 40 microliters DDH20 , let stand 1 minute, centrifuge 1 min</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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<div id='contentbox'>
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<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
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<b>Fast Protein Liquid Chromatography</b></a>
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<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
/*Materials:
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<ul class="a">
 +
  <li>- </li>
 +
  <li>- </li>
 +
  <li>- </li>
 +
  <li>- </li>
 +
  <li>- </li>
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</ul>*/
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<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Procedure:
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<ul class="a">
 +
  <li>- centrifuge cell lysate (balance with equal mass of water) for 60 minutes at 25000 rpm</li>
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  <li>- Wash FPLC column with equilibration column (X2) with  syringe</li>
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  <li>- Wash out FLPC system with equilibration buffer</li>
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  <li>- Wash affinity column with equilibration column with  syringe</li>
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  <li>- Wash affinity  column with cobalt acetate with syringe</li>
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  <li>- Wash off excess cobalt with syringe</li>
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  <li>- Connect affinity column to FPLC system</li>
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  <li>- Inject supernatan</li>
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  <li>- Wash FPLC column with equilibration column (X2) with syringe</li>
 
</ul>
 
</ul>
 
<br>
 
<br>

Revision as of 10:30, 18 September 2015