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> | ||
+ | </ul> | ||
+ | <br> | ||
+ | <br> | ||
+ | </div> | ||
+ | |||
+ | <div id='contentbox'> | ||
+ | <p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;"> | ||
+ | <b>Gel Extract</b></a> | ||
+ | <p style="font-size:24px;text-align:left;text-decoration: underline;"> | ||
+ | /*Materials: | ||
+ | <ul class="a"> | ||
+ | <li>- </li> | ||
+ | <li>- </li> | ||
+ | <li>- </li> | ||
+ | <li>- </li> | ||
+ | <li>- </li> | ||
+ | </ul>*/ | ||
+ | <p style="font-size:24px;text-align:left;text-decoration: underline;"> | ||
+ | Procedure: | ||
+ | <ul class="a"> | ||
+ | <li>- cut out gel portions with scalpel</li> | ||
+ | <li>- weigh gel slice in colorless tube. Add 3 volumes of QG buffer to 1 volume of gel</li> | ||
+ | <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 | ||
+ | </li> | ||
+ | <li>- After the gel dissolves completely, check color of the mixture is yellow (it may be orange or violet) | ||
+ | </li> | ||
+ | <li>- Add 10 µL of 3M sodium acetate (ph 5.0) to make solution more yellow (regardless of current solution color)</li> | ||
+ | <li>- The color of solution relates to ph indicator in Q3</li> | ||
+ | <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> | ||
+ | <li>- Place a Q1Aquick spin column in a provided 2ml collection tube</li> | ||
+ | <li>- Apply sample to spin column, centrifuge for 1 min to bind dna</li> | ||
+ | <li>- discard flow through</li> | ||
+ | <li>- wash with .75 mL buffer PE in column, centrifuge 1 minute</li> | ||
+ | <li>- Place column in a clean 1.5 microcentrifuge tube</li> | ||
+ | <li>- 40 microliters DDH20 , let stand 1 minute, centrifuge 1 min</li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | <br> | ||
+ | </div> | ||
+ | <div id='contentbox'> | ||
+ | <p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;"> | ||
+ | <b>Fast Protein Liquid Chromatography</b></a> | ||
+ | <p style="font-size:24px;text-align:left;text-decoration: underline;"> | ||
+ | /*Materials: | ||
+ | <ul class="a"> | ||
+ | <li>- </li> | ||
+ | <li>- </li> | ||
+ | <li>- </li> | ||
+ | <li>- </li> | ||
+ | <li>- </li> | ||
+ | </ul>*/ | ||
+ | <p style="font-size:24px;text-align:left;text-decoration: underline;"> | ||
+ | Procedure: | ||
+ | <ul class="a"> | ||
+ | <li>- centrifuge cell lysate (balance with equal mass of water) for 60 minutes at 25000 rpm</li> | ||
+ | <li>- Wash FPLC column with equilibration column (X2) with syringe</li> | ||
+ | <li>- Wash out FLPC system with equilibration buffer</li> | ||
+ | <li>- Wash affinity column with equilibration column with syringe</li> | ||
+ | <li>- Wash affinity column with cobalt acetate with syringe</li> | ||
+ | <li>- Wash off excess cobalt with syringe</li> | ||
+ | <li>- Connect affinity column to FPLC system</li> | ||
+ | <li>- Inject supernatan</li> | ||
+ | <li>- Wash FPLC column with equilibration column (X2) with syringe</li> | ||
</ul> | </ul> | ||
<br> | <br> |
Revision as of 10:30, 18 September 2015