Team:Marburg/Notebook:October
From 2013.igem.org
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<div class="aim"> | <div class="aim"> | ||
<span class="aim">Aim:</span> | <span class="aim">Aim:</span> | ||
- | <span class="aim-desc">Test of disruption method | + | <span class="aim-desc">Test of glass beads disruption method. |
</span> | </span> | ||
</div> | </div> | ||
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<!-- Cell disruption --> | <!-- Cell disruption --> | ||
<fieldset class="experiment sonification"> | <fieldset class="experiment sonification"> | ||
- | <legend><a name="soni">Cell disruption | + | <legend><a name="soni">Cell disruption by means of ball mill</a></legend> |
<div class="investigator"> | <div class="investigator"> | ||
<span class="inv">Investigator:</span> | <span class="inv">Investigator:</span> | ||
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<div class="aim"> | <div class="aim"> | ||
<span class="aim">Aim:</span> | <span class="aim">Aim:</span> | ||
- | <span class="aim-desc">Test of | + | <span class="aim-desc">Test of ball mill disruption method. |
</span> | </span> | ||
</div> | </div> | ||
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<li>Two steel balls were added → Tube was shock freezed again.</li> | <li>Two steel balls were added → Tube was shock freezed again.</li> | ||
<li>Cell disruption was carried out by 3 times shaking for 30 sec and another shock freezing.</li> | <li>Cell disruption was carried out by 3 times shaking for 30 sec and another shock freezing.</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </fieldset> | ||
+ | |||
+ | <!-- Cell disruption --> | ||
+ | <fieldset class="experiment sonification"> | ||
+ | <legend><a name="soni">Cell disruption by means of sonification</a></legend> | ||
+ | <div class="investigator"> | ||
+ | <span class="inv">Investigator:</span> | ||
+ | <span class="inv-names">Franzi</span> | ||
+ | </div> | ||
+ | <div class="aim"> | ||
+ | <span class="aim">Aim:</span> | ||
+ | <span class="aim-desc">Test of sonification disruption method. | ||
+ | </span> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p><ul class="digest"> | ||
+ | <li>4 ml cell culture was harvested (5 min centrifugation at full speed.</li> | ||
+ | <li>The Supernatent was discarded and the pellet frozen in liquid nitrogen.</li> | ||
+ | <li>The frozen pellet was resuspended in 0.68 ml IP buffer with 3.4 µl PIC and sonicated (48 cycles, 30 sec on, 30 sec off, 4°C, high).</li> | ||
+ | <li>Extract was centrifuged at 7000 rcf for 5 min, the supernatant was transferred in a new reaction tube.</li> | ||
+ | <li>1,7 µl supernatant of cell lysis were mixed with 350 µl 70 % TCA and incubated for 20 min on ice.</li> | ||
+ | <li>The mixture was centrifuged for 15 min at 20.000 rcf and the pellet washed twice with 1 ml acetone.</li> | ||
+ | <li>The pellet was dried at RT and resuspended in 60 µl 8M ureabuffer (15 min 60 °C).</li> | ||
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="exp-content"> | <div class="exp-content"> | ||
<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>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 | + | <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>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> | <p>10 µg of each sample were used for SDS-PAGE (Westernblot).</p> |
Revision as of 09:37, 28 October 2013