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Team:Bielefeld-Germany/Labjournal/June
From 2013.igem.org
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| + | <div class="cyt_frag1_headline"> | ||
| + | <a name="cyt_frag1"><span style="color:#ff6600">[Cytochromes]</span> Phusion 50ul for mtrCAB Fragment1</span></a> | ||
| + | </div> | ||
| + | <div class="cyt_frag1"> | ||
| + | <table border=1 style="float:left; padding-left:10px"> | ||
| + | <col width="150"> | ||
| + | <col width="50"> | ||
| + | <col width="50"> | ||
| + | <tr> <th>Reagent</th> <th>1x</th> <th>8x</th> </tr> | ||
| + | <tr> <td>Hf-Buffer 5x</td> <td align="right">10.0</td> <td align="right">88.0</td> </tr> | ||
| + | <tr> <td>dNTPs (10mM)</td> <td align="right">2.0</td> <td align="right">17.6</td> </tr> | ||
| + | <tr> <td>Primer1 (10mM)</td> <td align="right">1.0</td> <td align="right">8.8</td> </tr> | ||
| + | <tr> <td>Primer2 (10mM)</td> <td align="right">1.0</td> <td align="right">8.8</td> </tr> | ||
| + | <tr> <td>Template (16.5ng)</td> <td align="right">3.0</td> <td align="right">26.4</td> </tr> | ||
| + | <tr> <td>DMSO</td> <td align="right">4.0</td> <td align="right">35.2</td> </tr> | ||
| + | <tr> <td>Phusion (0.5u)</td> <td align="right">1.0</td> <td align="right">8.8</td> </tr> | ||
| + | <tr> <td>H2O</td> <td align="right">26.0</td> <td align="right">228.8</td> </tr> | ||
| + | </table> | ||
| + | |||
| + | <table border=1 style="float:left; margin-left:20px"> | ||
| + | <col width="50"> | ||
| + | <col width="50"> | ||
| + | <col width="50"> | ||
| + | <col width="50"> | ||
| + | <col width="50"> | ||
| + | <tr><th colspan="5">Program Phusion</th></tr> | ||
| + | <tr align="center"><td>98 °C</td><td>98 °C</td><td>68 °C</td><td>72 °C</td><td>72 °C</td></tr> | ||
| + | <tr align="center"><td>30s</td> <td>10s</td><td>30s</td> <td>1:35</td><td>6:00</td></tr> | ||
| + | <tr><td></td><td colspan="3" align="center">35 cycles</td><td></td></tr> | ||
| + | <tr><td colspan="5"> | ||
| + | <b><u>Notes:</u></b> | ||
| + | <br>Size: 1800bp | ||
| + | <br>Primer1: | ||
| + | <br>Primer2: | ||
| + | <br> | ||
| + | </td></tr> | ||
| + | </table> | ||
| + | |||
| + | |||
| + | <br><br><br><br><br><br><br><br><br><br><br><br><br> | ||
| + | </div> | ||
<h4><a href="https://2013.igem.org/Team:Bielefeld-Germany/Project/Cytochromes">Cytochromes</a></h4> | <h4><a href="https://2013.igem.org/Team:Bielefeld-Germany/Project/Cytochromes">Cytochromes</a></h4> | ||
Revision as of 13:17, 2 September 2013
June
Milesstones
1.Week
MFC
Mediators
Cytochromes
Biosafety
Porines
2.Week
MFC
Mediators
Cytochromes
Biosafety
Porines
3.Week
MFC
Mediators
| Reagent | 1x | 8x |
|---|---|---|
| Hf-Buffer 5x | 10.0 | 88.0 |
| dNTPs (10mM) | 2.0 | 17.6 |
| Primer1 (10mM) | 1.0 | 8.8 |
| Primer2 (10mM) | 1.0 | 8.8 |
| Template (16.5ng) | 3.0 | 26.4 |
| DMSO | 4.0 | 35.2 |
| Phusion (0.5u) | 1.0 | 8.8 |
| H2O | 26.0 | 228.8 |
| Program Phusion | ||||
|---|---|---|---|---|
| 98 °C | 98 °C | 68 °C | 72 °C | 72 °C |
| 30s | 10s | 30s | 1:35 | 6:00 |
| 35 cycles | ||||
|
Notes:
Size: 1800bp Primer1: Primer2: | ||||
Cytochromes
Cultivation of Shewanella oneidensis MR-1 in liquid LB medium at 30 °C
Isolation of genomic DNA from S. oneidensis
4-2006-451: Conc.:453.1 ng/µl OD260/280: 1.92 OD260/230:2.78
4-2006-452: Conc.:447.1 ng/µl OD260/280: 1.92 OD260/230:2.67
Dilution to PCR template: 4-2006-453: 5.5ng/µl
Amplification of the mtrCAB cluster with gradient PhusionPCR
Gradient: 55.8-56.7-57.8-59.1-60.4-61.7-62.9-63.9
Clear Bands at the expected 5.2kb
PCR-CleanUp of lane 2 and 5 with the Machery-Nagel-PCR-CleanUp-Kit
Lane2: 4-2106-451: 7.4 ng/µl
Lane5: 4-2106-451: 8.5 ng/µl
