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Team:Marburg:Project:Notebook
From 2014.igem.org
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<legend><a name="exp1.1">1.1 Overnight Preculture</a></legend> | <legend><a name="exp1.1">1.1 Overnight Preculture</a></legend> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
| - | <p> 2x5 mL LB medium were inoculated with 50 µL | + | <p> 2x5 mL LB medium were inoculated with 50 µL DH5α, aliquoted for a preculture and incubated overnight (16,5h) at 37 °C.</p> |
</div> | </div> | ||
</fieldset> | </fieldset> | ||
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<div class="exp-content"> | <div class="exp-content"> | ||
<!-- exp1.3 --> | <!-- exp1.3 --> | ||
| - | <p> | + | <p>250 mL were inoculated with 5 mL of preculture and incubated until they reached an OD of 0,5. After centrifugation, the cell pellet was washed with HTP buffer and resuspended in 3 mL HTP buffer. In the end, 225 µL DMSO was added to an end concentration of 6-7%. The 50 µL aliquots were frozen in N<sub>2</sub>(l) and stored at -80 °C. </p> |
</div> | </div> | ||
</fieldset> | </fieldset> | ||
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</h2> | </h2> | ||
<fieldset class="exp5"> | <fieldset class="exp5"> | ||
| - | <legend><a name="exp5.1">5.1 Testing Competence and Transformation of | + | <legend><a name="exp5.1">5.1 Testing Competence and Transformation of DH5α</a></legend> |
<div class="exp-content"> | <div class="exp-content"> | ||
| - | <p>1µ | + | <p>1 µL plasmid with a Amp-resistance was put on ice for 10 min. A heat shock was induced for 45 s at 42 °C. The sample was put on ice for another 10 min and then incubated in 200 µL LB for 1 h before it was plated onto LB-, ampicillin- and canamycin-plates.</p> |
</div> | </div> | ||
</fieldset> | </fieldset> | ||
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<legend><a name="exp7.1">7.1 <i>E. coli</i> DH5α with PSG1164: Overnight-cultures</a></legend> | <legend><a name="exp7.1">7.1 <i>E. coli</i> DH5α with PSG1164: Overnight-cultures</a></legend> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
| - | <p> | + | <p>5 mL LB-medium (with ampicillin) were inoculated with <i>E.coli</i> BL21 pLysS and grown overnight.</p> |
</div> | </div> | ||
</fieldset> | </fieldset> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td> | + | <td>ampicillin-plate</td> |
<td>no growth</td> | <td>no growth</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <td> | + | <td>ampicillin-plate 2</td> |
| - | <td>trafo | + | <td>trafo successful growth</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| + | <!-- <img src="" alt="" /> --> | ||
</div> | </div> | ||
</fieldset> | </fieldset> | ||
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<legend><a name="exp7.2">7.2 Inoculation</a></legend> | <legend><a name="exp7.2">7.2 Inoculation</a></legend> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
| - | <p>Inoculation of LB-plates with E.Coli | + | <p>Inoculation of LB-plates with <i>E.Coli</i> DH5α with PSG1164.</p> |
</div> | </div> | ||
</fieldset> | </fieldset> | ||
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<div class="exp-content"> | <div class="exp-content"> | ||
<!-- exp7.4 --> | <!-- exp7.4 --> | ||
| - | <P>Miniprep according to the miniprep kit protocol (Omega) with | + | <P>Miniprep according to the miniprep kit protocol (Omega) with 2x6 mL preculture.</P> |
<p>DNA-concentration:</p> | <p>DNA-concentration:</p> | ||
<ul class="miniprep"> | <ul class="miniprep"> | ||
| - | <li>1. 233,2 ng/µ | + | <li>1. 233,2 ng/µL</li> |
| - | <li>2. 261,3 ng/µ | + | <li>2. 261,3 ng/µL</li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<table width="100%" border="1"> | <table width="100%" border="1"> | ||
<tr> | <tr> | ||
| - | <th scope="col">[µ | + | <th scope="col">[µL]</th> |
<th scope="col">PSG1164 - uncut</th> | <th scope="col">PSG1164 - uncut</th> | ||
| - | <th scope="col">PSG1164 - | + | <th scope="col">PSG1164 - NcoI (20kU/mL)</th> |
<th scope="col">PSG1164 - EcoRI-HF</th> | <th scope="col">PSG1164 - EcoRI-HF</th> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<th scope="row">DNA</th> | <th scope="row">DNA</th> | ||
| - | <td>261,3 ng/µ | + | <td>261,3 ng/µL<br /> |
| - | ca. 1 µg → 3,5 µ | + | ca. 1 µg → 3,5 µL</td> |
| - | <td>261,3 ng/µ | + | <td>261,3 ng/µL<br /> |
| - | ca. 1 µg → 3,5 µ | + | ca. 1 µg → 3,5 µL</td> |
| - | <td>261,3 ng/µ | + | <td>261,3 ng/µL<br /> |
| - | ca. 1 µg → 3,5 µ | + | ca. 1 µg → 3,5 µL</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
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<td>10x Tango Buffer<br /> | <td>10x Tango Buffer<br /> | ||
1,5</td> | 1,5</td> | ||
| - | <td>10x | + | <td>10x Cutsmart<br /> |
1,5</td> | 1,5</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | <th scope="row"> | + | <th scope="row">H<sub>2</sub>O</th> |
<td>11,5</td> | <td>11,5</td> | ||
<td>9,5</td> | <td>9,5</td> | ||
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</tr> | </tr> | ||
</table> | </table> | ||
| - | <p>Incubate samples for 60 min at 37°C, then induce a heat shock for 2 min at 45°C. 5µ | + | <p>Incubate samples for 60 min at 37 °C, then induce a heat shock for 2 min at 45 °C. 5 µL marker and 10 µL of the samples are loaded onto a 1% agarose gel.</p> |
</div> | </div> | ||
<div class="exp-results"> | <div class="exp-results"> | ||
<h3>Results:</h3> | <h3>Results:</h3> | ||
<!-- exp7.7 --> | <!-- exp7.7 --> | ||
| - | <img src="https://static.igem.org/mediawiki/2014/ | + | <img src="https://static.igem.org/mediawiki/2014/3/34/MR_20140327_restriction_pSG1164_NcoI_and_EcoRI.jpg" width="30%" /> |
| + | <p>The plasmid digested with <i>Nco</i>I showed two bands, what means, it had two restriction sites. pSG1164 digested with <i>Eco</i>RI looked like it was linearized.</p> | ||
</div> | </div> | ||
</fieldset> | </fieldset> | ||
Latest revision as of 11:45, 16 October 2014
