Team:Marburg/Notebook:October
From 2013.igem.org
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<span class="aim">Aim:</span> | <span class="aim">Aim:</span> | ||
- | <span class="aim-desc">Quantification of P<sub>fcpB</sub> → Cell disruption, protein precipitation and SDS-PAGE.</span> | + | <span class="aim-desc">Quantification of the light-inducible promoter P<sub>fcpB</sub> (<html><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1071003" target="_blank">BBa_K1071003</a></html> → Cell disruption, protein precipitation and SDS-PAGE.</span> |
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- | <p>10 ml of cultures grown under different light | + | <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>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> |
Revision as of 17:28, 27 October 2013