Team:Marburg:Project:Notebook

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Notebook: March
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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>
 +
<div class="exp-content">
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<p> 2x5 mL LB medium were inoculated with 50 &micro;L DH5&alpha;, aliquoted  for a preculture and incubated overnight (16,5h) at 37 &deg;C.</p>
 +
    </div>
 +
</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">
 +
    <legend><a name="exp1.2">1.2 Main Culture</a></legend>
 +
<div class="exp-content">
 +
    <!-- exp1.3 -->
 +
<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 &micro;L DMSO was added to an end concentration of 6-7%. The 50 &micro;L aliquots were frozen in N<sub>2</sub>(l) and stored at -80 &deg;C. </p>
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</div>
 +
</fieldset>
 +
</div>
 +
 +
<!-- 26.03.14 -->
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 +
<div class="notebooky-entry">
 +
<h2 class="title">
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<a name="26.03.2014">26.03.2014</a>
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</h2>
 +
<fieldset class="exp5">
 +
    <legend><a name="exp5.1">5.1 Testing Competence and Transformation of DH5&alpha;</a></legend>
 +
    <div class="exp-content">
 +
<p>1 &micro;L plasmid with a Amp-resistance was put on ice for 10 min. A heat shock was induced for 45 s at 42 &deg;C. The sample was put on ice for another 10 min and then incubated in 200 &micro;L LB for 1 h before it was plated onto LB-, ampicillin- and canamycin-plates.</p>
 +
    </div>
 +
</fieldset>
 +
 +
<fieldset class="exp7">
 +
    <legend><a name="exp7.1">7.1 <i>E. coli</i> DH5&alpha; with PSG1164: Overnight-cultures</a></legend>
 +
<div class="exp-content">
 +
<p>5 mL LB-medium (with ampicillin) were inoculated with <i>E.coli</i> BL21 pLysS and grown overnight.</p>
 +
    </div>
 +
</fieldset>
 +
</div>
 +
 +
<!-- 27.03.14 -->
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 +
<div class="notebooky-entry">
 +
<h2 class="title">
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<a name="27.03.2014">27.03.2014</a>
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</h2>
 +
<fieldset class="exp5">
 +
    <legend><a name="exp5.2">5.2 Results</a></legend>
 +
<div class="exp-content">
 +
<p>Plates from 26.03.2014</p>
 +
<table class="results" width="40%" border="1">
 +
  <tr>
 +
    <td width="50%">LB-plate</td>
 +
    <td width="50%">growth</td>
 +
      </tr>
 +
  <tr>
 +
    <td>canamycin-plate</td>
 +
    <td>no growth</td>
 +
      </tr>
 +
  <tr>
 +
    <td>ampicillin-plate</td>
 +
    <td>no growth</td>
 +
      </tr>
 +
  <tr>
 +
    <td>ampicillin-plate 2</td>
 +
    <td>trafo successful growth</td>
 +
      </tr>
 +
  </table>
 +
<!-- <img src="" alt="" /> -->
 +
    </div>
 +
</fieldset>
 +
 +
<fieldset class="exp7">
 +
    <legend><a name="exp7.2">7.2 Inoculation</a></legend>
 +
<div class="exp-content">
 +
<p>Inoculation of LB-plates with <i>E.Coli</i> DH5&alpha; with PSG1164.</p>
 +
    </div>
 +
</fieldset>
 +
<fieldset class="exp7">
 +
    <legend><a name="exp7.3">7.3 Miniprep</a></legend>
 +
<div class="exp-content">
 +
    <!-- exp7.4 -->
 +
    <P>Miniprep according to the miniprep kit protocol (Omega) with 2x6 mL 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>
 +
    <th scope="col">Tested enzyme</th>
 +
    <th scope="col">Compatible with Ncol</th>
 +
    <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>
 +
        <td>50</td>
 +
        <td>50*</td>
 +
      </tr>
 +
      <tr>
 +
        <td>ApaI / HF</td>
 +
        <td>100</td>
 +
        <td>100</td>
 +
      </tr>
 +
      <tr>
 +
        <td>XhoI / HF</td>
 +
        <td>100</td>
 +
        <td>100</td>
 +
      </tr>
 +
      <tr>
 +
        <td>SalI / HF</td>
 +
        <td>10</td>
 +
        <td>10/100</td>
 +
      </tr>
 +
      <tr>
 +
        <td>ClaI / HF</td>
 +
        <td>100</td>
 +
        <td>100</td>
 +
      </tr>
 +
      <tr>
 +
        <td>HindIII / HF</td>
 +
        <td>50*/100</td>
 +
        <td>50/100</td>
 +
      </tr>
 +
      <tr>
 +
        <td>EcoRV / HF</td>
 +
        <td>10*/100</td>
 +
        <td>10/100</td>
 +
      </tr>
 +
      <tr>
 +
        <td>EcoRI / HF</td>
 +
        <td>50*/100</td>
 +
        <td>50*100</td>
 +
      </tr>
 +
      <tr>
 +
        <td>PstI / HF</td>
 +
        <td>50*/100</td>
 +
        <td>50*100</td>
 +
      </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 - NcoI (20kU/mL)</th>
 +
        <th scope="col">PSG1164 - EcoRI-HF</th>
 +
        </tr>
 +
      <tr>
 +
        <th scope="row">DNA</th>
 +
        <td>261,3 ng/&micro;L<br />
 +
          ca. 1 &micro;g &rarr; 3,5 &micro;L</td>
 +
        <td>261,3 ng/&micro;L<br />
 +
    ca. 1 &micro;g &rarr; 3,5 &micro;L</td>
 +
        <td>261,3 ng/&micro;L<br />
 +
    ca. 1 &micro;g &rarr; 3,5 &micro;L</td>
 +
        </tr>
 +
      <tr>
 +
        <th scope="row">Enzyme 1</th>
 +
        <td>-</td>
 +
        <td>Ncol<br />
 +
          0,5</td>
 +
        <td>-</td>
 +
        </tr>
 +
      <tr>
 +
        <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 Cutsmart<br />
 +
          1,5</td>
 +
        </tr>
 +
      <tr>
 +
        <th scope="row">H<sub>2</sub>O</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 a 1% agarose gel.</p>
 +
    </div>
 +
<div class="exp-results">
 +
    <h3>Results:</h3>
 +
    <!-- exp7.7 -->
 +
<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>
 +
</fieldset>
 +
</div>
 +
 +
</html>
{{Team:Marburg/Template:End}}
{{Team:Marburg/Template:End}}

Latest revision as of 11:45, 16 October 2014

Notebook: March

17.03.2014

1.1 Overnight Preculture

2x5 mL LB medium were inoculated with 50 µL DH5α, aliquoted for a preculture and incubated overnight (16,5h) at 37 °C.

18.03.2014

1.2 Main Culture

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 N2(l) and stored at -80 °C.

26.03.2014

5.1 Testing Competence and Transformation of DH5α

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.

7.1 E. coli DH5α with PSG1164: Overnight-cultures

5 mL LB-medium (with ampicillin) were inoculated with E.coli BL21 pLysS and grown overnight.

27.03.2014

5.2 Results

Plates from 26.03.2014

LB-plate growth
canamycin-plate no growth
ampicillin-plate no growth
ampicillin-plate 2 trafo successful growth
7.2 Inoculation

Inoculation of LB-plates with E.Coli DH5α with PSG1164.

7.3 Miniprep

Miniprep according to the miniprep kit protocol (Omega) with 2x6 mL 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 - NcoI (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 Cutsmart
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. 5 µL marker and 10 µL of the samples are loaded onto a 1% agarose gel.

Results:

The plasmid digested with NcoI showed two bands, what means, it had two restriction sites. pSG1164 digested with EcoRI looked like it was linearized.

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