Team:Marburg/Notebook:October
From 2013.igem.org
(Difference between revisions)
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+ | </div> | ||
+ | |||
+ | <div class="notebooky-entry"> | ||
+ | <h2 class="title"> | ||
+ | <a name="21-10-2013">21.10.2013</a> | ||
+ | </h2> | ||
+ | |||
+ | <!-- Cell disruption --> | ||
+ | <fieldset class="experiment sonification"> | ||
+ | <legend><a name="soni">Cell disruption by means of glass beads</a></legend> | ||
+ | <div class="investigator"> | ||
+ | <span class="inv">Investigator:</span> | ||
+ | <span class="inv-names">Dominik</span> | ||
+ | </div> | ||
+ | <div class="aim"> | ||
+ | <span class="aim">Aim:</span> | ||
+ | <span class="aim-desc">Test of disruption method: Glass beads. | ||
+ | </span> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p><ul class="digest"> | ||
+ | <li>4 ml cell culture was harvested.</li> | ||
+ | <li>Pellet was dissolved in 1 mL PBS.</li> | ||
+ | <li>0.5 ml glass beads were added.</li> | ||
+ | <li>Tubes were shaken for 3 min → color shift from brown to green.</li> | ||
+ | <li>TCA precipitation was performed.</li> | ||
+ | </ul> | ||
+ | <p>10 ml of cultures grown under different light conditions (green, blue, red, full light spectrum and darkness) were harvested in 15 ml Falcon-tubes (5 min centrifugation at full speed). Supernatants were discarded and pellets frozen in liquid nitrogen.</p> | ||
+ | <p>Frozen cell-pellets were resuspended in 1,7 ml IP-buffer with 8,5 µl PIC and sonicated (48 cycles, 30 sec on, 30 sec off, 4°C, high). Extract was centrifuged at 7000 rcf for 5 min, the supernatant was transferred in a new reaction tube.</p> | ||
+ | <p>1,7 µl supernatant of cell lysis were mixed with 350 µl 70 % TCA and incubated for 20 min on ice. The mixture was centrifuged for 15 min at 20.000 rcf and the pellet washed twice with 1 ml acetone. The pellet was dried at RT and resuspended in 60 µl 8M ureabuffer (15 min 60 °C).</p> | ||
+ | <p>10 µg of each sample were used for SDS-PAGE (Westernblot).</p> | ||
+ | </div> | ||
+ | </fieldset> | ||
</div> | </div> | ||
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<legend><a name="soni">Sonification</a></legend> | <legend><a name="soni">Sonification</a></legend> | ||
<div class="investigator"> | <div class="investigator"> | ||
- | <span class="inv"> | + | <span class="inv">Investigators:</span> |
<span class="inv-names">Franzi, Marco</span> | <span class="inv-names">Franzi, Marco</span> | ||
</div> | </div> |
Revision as of 21:45, 27 October 2013