Team:Marburg:Project:Notebook

From 2014.igem.org

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<!-- 17.03.14 -->
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<div class="notebooky-entry">
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<h2 class="title">
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<a name="17.03.2014">17.03.2014</a>
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</h2>
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<fieldset class="exp1">
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    <legend><a name="exp1.1">1.1 Overnight Preculture</a></legend>
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<div class="exp-content">
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<p> 2x5ml LB medium were inoculated with 50&micro;l DH5Alpha, aliquoted  for a preculture and incubated overnight (16,5h) at 37&deg;C.</p>
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    </div>
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</fieldset>
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</div>
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<!-- 18.03.14 -->
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<div class="notebooky-entry">
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<h2 class="title">
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<a name="18.03.2014">18.03.2014</a>
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</h2>
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<fieldset class="exp1">
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    <legend><a name="exp1.2">1.2 Main Culture</a></legend>
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<div class="exp-content">
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    <!-- exp1.3 -->
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<p>250ml were inoculated with 5ml 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 3ml HTP buffer. In the end, 225&micro;l DMSO was added to an end concenctration of 6-7%. The 50&micro;l aliquots were frozen in N2(l) and stored at -80&deg;C. </p>
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</div>
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</fieldset>
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</div>
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<!-- 26.03.14 -->
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<div class="notebooky-entry">
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<h2 class="title">
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<a name="26.03.2014">26.03.2014</a>
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</h2>
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<fieldset class="exp5">
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    <legend><a name="exp5.1">5.1 Testing Competence and Transformation of DH5Alpha</a></legend>
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    <div class="exp-content">
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<p>1&micro;l plasmid with a amp-resistence was put on ice for 10min. A heat shock was induced for 45s at 42°C. The sample was put on ice for another 10min and then incubated in 200&micro;l LB for 1h before it was plated onto LB-, ampicilin- and canamycin-plates.</p>
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    </div>
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</fieldset>
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<fieldset class="exp7">
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    <legend><a name="exp7.1">7.1 E.coli DH5Alpha with PSG1164: Overnight-cultures</a></legend>
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<div class="exp-content">
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<p>50ml LB-medium (with ampicilin) were inoculated with E.coli BL21 PlyS and grown overnight.</p>
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    </div>
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</fieldset>
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</div>
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 +
<!-- 27.03.14 -->
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 +
<div class="notebooky-entry">
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<h2 class="title">
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<a name="27.03.2014">27.03.2014</a>
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</h2>
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<fieldset class="exp5">
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    <legend><a name="exp5.2">5.2 Results</a></legend>
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<div class="exp-content">
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<p>Plates from 26.03.2014</p>
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<table class="results" width="40%" border="1">
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  <tr>
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    <td width="50%">LB-plate</td>
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    <td width="50%">growth</td>
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      </tr>
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  <tr>
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    <td>canamycin-plate</td>
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    <td>no growth</td>
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      </tr>
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  <tr>
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    <td>ampicilin-plate</td>
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    <td>no growth</td>
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      </tr>
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  <tr>
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    <td>ampicilin-plate 2</td>
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    <td>trafo successfull growth</td>
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      </tr>
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  </table>
 +
    </div>
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</fieldset>
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 +
<fieldset class="exp7">
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    <legend><a name="exp7.2">7.2 Inoculation</a></legend>
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<div class="exp-content">
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<p>Inoculation of LB-plates with E.Coli DH5Alpha with PSG1164.</p>
 +
    </div>
 +
</fieldset>
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<fieldset class="exp7">
 +
    <legend><a name="exp7.3">7.3 Miniprep</a></legend>
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<div class="exp-content">
 +
    <!-- exp7.4 -->
 +
    <P>Miniprep according to the miniprep kit protocol (Omega) with 2x6ml preculture.</P>
 +
        <p>DNA-concentration:</p>
 +
        <ul class="miniprep">
 +
<li>1. 233,2 ng/&micro;l</li>
 +
            <li>2. 261,3 ng/&micro;l</li>
 +
</ul>
 +
    </div>
 +
</fieldset>
 +
<fieldset class="exp7">
 +
    <legend><a name="exp7.5">7.5 Test Digestion</a></legend>
 +
<div class="exp-content">
 +
    <table width="100%" border="1">
 +
      <tr>
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    <th scope="col">Tested enzyme</th>
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    <th scope="col">Compatible with Ncol</th>
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    <th scope="col">Compatible with Ncol-HF</th>
 +
      </tr>
 +
      <tr>
 +
        <td>AvrII</td>
 +
        <td>100</td>
 +
        <td>100</td>
 +
      </tr>
 +
      <tr>
 +
        <td>KpnI / HF</td>
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        <td>50</td>
 +
        <td>50*</td>
 +
      </tr>
 +
      <tr>
 +
        <td>ApaI / HF</td>
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        <td>100</td>
 +
        <td>100</td>
 +
      </tr>
 +
      <tr>
 +
        <td>XhoI / HF</td>
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        <td>100</td>
 +
        <td>100</td>
 +
      </tr>
 +
      <tr>
 +
        <td>SalI / HF</td>
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        <td>10</td>
 +
        <td>10/100</td>
 +
      </tr>
 +
      <tr>
 +
        <td>ClaI / HF</td>
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        <td>100</td>
 +
        <td>100</td>
 +
      </tr>
 +
      <tr>
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        <td>HindIII / HF</td>
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        <td>50*/100</td>
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        <td>50/100</td>
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      </tr>
 +
      <tr>
 +
        <td>EcoRV / HF</td>
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        <td>10*/100</td>
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        <td>10/100</td>
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      </tr>
 +
      <tr>
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        <td>EcoRI / HF</td>
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        <td>50*/100</td>
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        <td>50*100</td>
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      </tr>
 +
      <tr>
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        <td>PstI / HF</td>
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        <td>50*/100</td>
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        <td>50*100</td>
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      </tr>
 +
    </table>
 +
    </div>
 +
</fieldset>
 +
<fieldset class="exp7">
 +
    <legend><a name="exp7.6">7.6 Digestion of PSG1164</a></legend>
 +
<div class="exp-content">   
 +
<h3>Digestion scheme:</h3>
 +
    <table width="100%" border="1">
 +
      <tr>
 +
        <th scope="col">[&micro;l]</th>
 +
        <th scope="col">PSG1164 - uncut</th>
 +
        <th scope="col">PSG1164 - NcolI (20kU/ml)</th>
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        <th scope="col">PSG1164 - EcoRI-HF</th>
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        </tr>
 +
      <tr>
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        <th scope="row">DNA</th>
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        <td>261,3 ng/&micro;l<br />
 +
          ca. 1 &micro;g &rarr; 3,5 &micro;l</td>
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        <td>261,3 ng/&micro;l<br />
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    ca. 1 &micro;g &rarr; 3,5 &micro;l</td>
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        <td>261,3 ng/&micro;l<br />
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    ca. 1 &micro;g &rarr; 3,5 &micro;l</td>
 +
        </tr>
 +
      <tr>
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        <th scope="row">Enzyme 1</th>
 +
        <td>-</td>
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        <td>Ncol<br />
 +
          0,5</td>
 +
        <td>-</td>
 +
        </tr>
 +
      <tr>
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        <th scope="row">Enzyme 2</th>
 +
        <td>-</td>
 +
        <td>-</td>
 +
        <td>EcoRI-HF<br />
 +
          0,5</td>
 +
        </tr>
 +
      <tr>
 +
        <th scope="row">Buffer</th>
 +
        <td>-</td>
 +
        <td>10x Tango Buffer<br />
 +
          1,5</td>
 +
        <td>10x Cutsmarkt<br />
 +
          1,5</td>
 +
        </tr>
 +
      <tr>
 +
        <th scope="row">H2O</th>
 +
        <td>11,5</td>
 +
        <td>9,5</td>
 +
        <td>9,5</td>
 +
        </tr>
 +
      <tr>
 +
        <th scope="row">Total Volume</th>
 +
        <td>15</td>
 +
        <td>15</td>
 +
        <td>15</td>
 +
        </tr>
 +
      <tr>
 +
        <th scope="row">Loading Dye (6x)</th>
 +
        <td>2,5</td>
 +
        <td>2,5</td>
 +
        <td>2,5</td>
 +
        </tr>
 +
    </table>
 +
<p>Incubate samples for 60 min at 37&deg;C, then induce a heat shock for 2 min at 45&deg;C. 5&micro;l marker and 10&micro;l of the samples are loaded onto the SDS-gel.</p>
 +
    </div>
 +
<div class="exp-results">
 +
    <h3>Results:</h3>
 +
    <!-- exp7.7 -->
 +
<img src="https://static.igem.org/mediawiki/2014/5/51/2014-03-07_PSG1164.jpg" width="30%" />
 +
    </div>
 +
</fieldset>
 +
</div>
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 +
</html>
{{Team:Marburg/Template:End}}
{{Team:Marburg/Template:End}}

Revision as of 15:31, 12 October 2014

Notebook: March

17.03.2014

1.1 Overnight Preculture

2x5ml LB medium were inoculated with 50µl DH5Alpha, aliquoted for a preculture and incubated overnight (16,5h) at 37°C.

18.03.2014

1.2 Main Culture

250ml were inoculated with 5ml 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 3ml HTP buffer. In the end, 225µl DMSO was added to an end concenctration of 6-7%. The 50µl aliquots were frozen in N2(l) and stored at -80°C.

26.03.2014

5.1 Testing Competence and Transformation of DH5Alpha

1µl plasmid with a amp-resistence was put on ice for 10min. A heat shock was induced for 45s at 42°C. The sample was put on ice for another 10min and then incubated in 200µl LB for 1h before it was plated onto LB-, ampicilin- and canamycin-plates.

7.1 E.coli DH5Alpha with PSG1164: Overnight-cultures

50ml LB-medium (with ampicilin) were inoculated with E.coli BL21 PlyS and grown overnight.

27.03.2014

5.2 Results

Plates from 26.03.2014

LB-plate growth
canamycin-plate no growth
ampicilin-plate no growth
ampicilin-plate 2 trafo successfull growth
7.2 Inoculation

Inoculation of LB-plates with E.Coli DH5Alpha with PSG1164.

7.3 Miniprep

Miniprep according to the miniprep kit protocol (Omega) with 2x6ml preculture.

DNA-concentration:

  • 1. 233,2 ng/µl
  • 2. 261,3 ng/µl
7.5 Test Digestion
Tested enzyme Compatible with Ncol Compatible with Ncol-HF
AvrII 100 100
KpnI / HF 50 50*
ApaI / HF 100 100
XhoI / HF 100 100
SalI / HF 10 10/100
ClaI / HF 100 100
HindIII / HF 50*/100 50/100
EcoRV / HF 10*/100 10/100
EcoRI / HF 50*/100 50*100
PstI / HF 50*/100 50*100
7.6 Digestion of PSG1164

Digestion scheme:

[µl] PSG1164 - uncut PSG1164 - NcolI (20kU/ml) PSG1164 - EcoRI-HF
DNA 261,3 ng/µl
ca. 1 µg → 3,5 µl		 261,3 ng/µl
ca. 1 µg → 3,5 µl		 261,3 ng/µl
ca. 1 µg → 3,5 µl
Enzyme 1 - Ncol
0,5		 -
Enzyme 2 - - EcoRI-HF
0,5
Buffer - 10x Tango Buffer
1,5		 10x Cutsmarkt
1,5
H2O 11,5 9,5 9,5
Total Volume 15 15 15
Loading Dye (6x) 2,5 2,5 2,5

Incubate samples for 60 min at 37°C, then induce a heat shock for 2 min at 45°C. 5µl marker and 10µl of the samples are loaded onto the SDS-gel.

Results:

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