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">Characterization of the light-inducible promotor P<sub>fcpB</sub> (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1071003 | + | <span class="aim-desc">Characterization of the light-inducible promotor P<sub>fcpB</sub> (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1071003">BBa_K1071003</a>).</span> |
</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
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<div class="aim"> | <div class="aim"> | ||
<span class="aim">Aim:</span> | <span class="aim">Aim:</span> | ||
- | <span class="aim-desc">Characterization of the light-inducible promotor P<sub>fcpB</sub> (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1071003 | + | <span class="aim-desc">Characterization of the light-inducible promotor P<sub>fcpB</sub> (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1071003">BBa_K1071003</a>).</span> |
</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
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<div class="aim"> | <div class="aim"> | ||
<span class="aim">Aim:</span> | <span class="aim">Aim:</span> | ||
- | <span class="aim-desc">Quantification of the light-inducible promoter P<sub>fcpB</sub> (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1071003 | + | <span class="aim-desc">Quantification of the light-inducible promoter P<sub>fcpB</sub> (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1071003">BBa_K1071003</a>)<br> → Cell disruption, protein precipitation and SDS-PAGE. |
</span> | </span> | ||
</div> | </div> | ||
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<li>Blocking: Incubate in 7 % milk powder in TBST buffer for 1 h at RT</li> | <li>Blocking: Incubate in 7 % milk powder in TBST buffer for 1 h at RT</li> | ||
<li>Wash three times for 10 min with TBST buffer at RT</li> | <li>Wash three times for 10 min with TBST buffer at RT</li> | ||
- | <li>Add the first antibody: | + | <li>Add the first antibody: goat anti-GFP (1:2000) in TBS with 0.02% NaN3 for 1.5 h at RT</li> |
- | <li>Add the second antiboy: anti-goat | + | <li>Add the second antiboy: anti-goat 800 (1:10000) in TBS buffer for 1 h at RT</li> |
<li>Wash two times for 10 min with TBST buffer at RT</li> | <li>Wash two times for 10 min with TBST buffer at RT</li> | ||
<li>Wash a last time for 10 min with TBS buffer at RT </li> | <li>Wash a last time for 10 min with TBS buffer at RT </li> | ||
- | <li>Detect with 1 | + | <li>Detect with scanner at 800 nm</li> |
+ | <li>Add the first antibody: mouse anti-tubulin (1:2000) in TBS with 0.02% NaN3 for 1.5 h at RT</li> | ||
+ | <li>Add the second antiboy: anti-mouse 800 (1:10000) in TBS buffer for 1 h at RT</li> | ||
+ | <li>Wash two times for 10 min with TBST buffer at RT</li> | ||
+ | <li>Wash a last time for 10 min with TBS buffer at RT </li> | ||
+ | <li>Detect with scanner at 800 nm</li> | ||
</ul> | </ul> | ||
+ | <p></p> | ||
<table class="gel pcr"> | <table class="gel pcr"> | ||
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<thead> | <thead> | ||
<tr> | <tr> | ||
- | <th colspan="2" class="title"> | + | <th colspan="2" class="title">Western blot</th> |
</tr> | </tr> | ||
</thead> | </thead> | ||
Line 857: | Line 863: | ||
<colgroup> | <colgroup> | ||
<col width="9%" /> | <col width="9%" /> | ||
+ | <col width="3%" /> | ||
+ | <col width="24%" /> | ||
<col width="2%" /> | <col width="2%" /> | ||
- | <col width=" | + | <col width="62%" /> |
- | + | ||
- | + | ||
</colgroup> | </colgroup> | ||
Line 875: | Line 881: | ||
<td>Darkness</td> | <td>Darkness</td> | ||
<th> </th> | <th> </th> | ||
- | <td> | + | <td>27 kDa (GFP) + 55 kDa (tubulin)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>3</td> | <td>3</td> | ||
<th> </th> | <th> </th> | ||
- | <td> | + | <td>Blue light</td> |
<th> </th> | <th> </th> | ||
- | <td> | + | <td>27 kDa (GFP) + 55 kDa (tubulin)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>4</td> | <td>4</td> | ||
<th> </th> | <th> </th> | ||
- | <td> | + | <td>Green light</td> |
<th> </th> | <th> </th> | ||
- | <td> | + | <td>27 kDa (GFP) + 55 kDa (tubulin)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>5</td> | <td>5</td> | ||
<th> </th> | <th> </th> | ||
- | <td> | + | <td>Red light</td> |
<th> </th> | <th> </th> | ||
- | <td> | + | <td>27 kDa (GFP) + 55 kDa (tubulin)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>6</td> | <td>6</td> | ||
<th> </th> | <th> </th> | ||
- | <td> | + | <td>Control</td> |
<th> </th> | <th> </th> | ||
- | <td> | + | <td>55 kDa (tubulin) + 80 kDa ("GFP")</td> |
</tr> | </tr> | ||
+ | </table> | ||
+ | <p><b>Lower gel</b></p> | ||
+ | <table> | ||
+ | <colgroup> | ||
+ | <col width="9%" /> | ||
+ | <col width="3%" /> | ||
+ | <col width="24%" /> | ||
+ | <col width="2%" /> | ||
+ | <col width="62%" /> | ||
+ | </colgroup> | ||
+ | |||
+ | <thead> | ||
+ | <th>Lane</th> | ||
+ | <th> </th> | ||
+ | <th>Content</th> | ||
+ | <th> </th> | ||
+ | <th>Expectations</th> | ||
+ | </thead> | ||
<tr> | <tr> | ||
- | <td> | + | <td>8</td> |
<th> </th> | <th> </th> | ||
- | <td> | + | <td>Light</td> |
<th> </th> | <th> </th> | ||
- | <td> | + | <td>27 kDa (GFP) + 55 kDa (tubulin)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td> | + | <td>9</td> |
<th> </th> | <th> </th> | ||
- | <td> | + | <td>Darkness</td> |
<th> </th> | <th> </th> | ||
- | <td> | + | <td>27 kDa (GFP) + 55 kDa (tubulin)</td> |
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10</td> | ||
+ | <th> </th> | ||
+ | <td>Blue light</td> | ||
+ | <th> </th> | ||
+ | <td>27 kDa (GFP) + 55 kDa (tubulin)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>11</td> | ||
+ | <th> </th> | ||
+ | <td>Green light</td> | ||
+ | <th> </th> | ||
+ | <td>27 kDa (GFP) + 55 kDa (tubulin)</td> | ||
+ | </tr><tr> | ||
+ | <td>12</td> | ||
+ | <th> </th> | ||
+ | <td>Red light</td> | ||
+ | <th> </th> | ||
+ | <td>27 kDa (GFP) + 55 kDa (tubulin)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>13</td> | ||
+ | <th> </th> | ||
+ | <td>Control</td> | ||
+ | <th> </th> | ||
+ | <td>55 kDa (tubulin) + 80 kDa ("GFP")</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
Line 925: | Line 976: | ||
</table> | </table> | ||
- | <p>Result: | + | <p>Result: All samples showed the expexted bands. For a detailed discussion have a look at our <a href="https://2013.igem.org/Team:Marburg/Project:lightcontrol">light control</a> section.</p> |
</div> | </div> |
Latest revision as of 22:50, 28 October 2013