Team:Marburg:Project:Notebook

From 2014.igem.org

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<div class="exp-content">
<div class="exp-content">
     <!-- exp1.3 -->
     <!-- exp1.3 -->
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<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 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>
+
<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>
</fieldset>
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     <legend><a name="exp5.1">5.1 Testing Competence and Transformation of DH5&alpha;</a></legend>
     <legend><a name="exp5.1">5.1 Testing Competence and Transformation of DH5&alpha;</a></legend>
     <div class="exp-content">
     <div class="exp-content">
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<p>1 &micro;L plasmid with a amp-resistence 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 1h before it was plated onto LB-, ampicilin- and canamycin-plates.</p>
+
<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>
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     <legend><a name="exp7.1">7.1 <i>E. coli</i> DH5&alpha; with PSG1164: Overnight-cultures</a></legend>
     <legend><a name="exp7.1">7.1 <i>E. coli</i> DH5&alpha; with PSG1164: Overnight-cultures</a></legend>
<div class="exp-content">
<div class="exp-content">
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<p>50 mL LB-medium (with ampicilin) were inoculated with <i>E.coli</i> BL21 PlyS and grown overnight.</p>
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<p>5 mL LB-medium (with ampicillin) were inoculated with <i>E.coli</i> BL21 pLysS and grown overnight.</p>
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      </tr>
      </tr>
  <tr>
  <tr>
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    <td>ampicilin-plate</td>
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    <td>ampicillin-plate</td>
    <td>no growth</td>
    <td>no growth</td>
      </tr>
      </tr>
  <tr>
  <tr>
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    <td>ampicilin-plate 2</td>
+
    <td>ampicillin-plate 2</td>
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    <td>trafo successfull growth</td>
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    <td>trafo successful growth</td>
      </tr>
      </tr>
  </table>
  </table>
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<!-- <img src="" alt="" /> -->
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         <th scope="col">[&micro;L]</th>
         <th scope="col">[&micro;L]</th>
         <th scope="col">PSG1164 - uncut</th>
         <th scope="col">PSG1164 - uncut</th>
-
         <th scope="col">PSG1164 - NcolI (20kU/mL)</th>
+
         <th scope="col">PSG1164 - NcoI (20kU/mL)</th>
         <th scope="col">PSG1164 - EcoRI-HF</th>
         <th scope="col">PSG1164 - EcoRI-HF</th>
         </tr>
         </tr>
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         <td>10x Tango Buffer<br />
         <td>10x Tango Buffer<br />
           1,5</td>
           1,5</td>
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         <td>10x Cutsmarkt<br />
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         <td>10x Cutsmart<br />
           1,5</td>
           1,5</td>
         </tr>
         </tr>
       <tr>
       <tr>
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         <th scope="row">H2O</th>
+
         <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>
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<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>
+
<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>
<div class="exp-results">
<div class="exp-results">
     <h3>Results:</h3>
     <h3>Results:</h3>
     <!-- exp7.7 -->
     <!-- exp7.7 -->
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<img src="https://static.igem.org/mediawiki/2014/5/51/2014-03-07_PSG1164.jpg" width="30%" />
+
<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

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.