Team:Groningen/Labwork/26 June 2013

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<div class="mainContent">
<div class="mainContent">
      
      
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<h2>Mirjam<h2>
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<h2>Mirjam</h2>
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<br/>Extraction of the genomic DNA of ''B. subtilis'' strain 168.
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Did a <a href="https://2013.igem.org/Team:Groningen/protocols/Genomic_DNA_extraction"><FONT COLOR="black"><b>genomic DNA extraction</b></FONT></a> of <i>B. subtilis</i> strain 168. Two tubes are obtained from this with a concentration of 66ng/&micro;l and 67ng/&micro;l.
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Diluting the primers (to 100 mM and 10 mM)
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<p>The primers for our main subject, silk secretion, arrived. The primers are diluted to a concentration of 10mM.
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<br/>Diluting the dNTPs to a concentration of 10 mM.  
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<br>To run a PCR, dNTPs are necessary in a concentration of 10mM, so this dilution is also made.
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A PCR reaction mix is made containing the following compounds:
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<p>A <a href="https://2013.igem.org/Team:Groningen/protocols/PCR"><FONT COLOR="black"><b>PCR reaction</b></FONT></a> mix is made for the four different signal sequences.
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<br/>5x Buffer HF  1x
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<br/>dNTPs          200 µM each
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<br/>primer F      1 µM
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<br/>primer R      1 µM
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<br/>temp DNA      6.7 ng
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<br/>phusion pol.  0.02 U/µl
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<br/>MQ water is added to get the wanted volume.
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<br>
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<table border="1">
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  <tr>
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    <th>signal sequence</th>
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    <th>T<sub>m</sub>-5</th>
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    <th>size</th>
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  </tr>
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Run a PCR for the four different signal sequences.
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  <tr>
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<br/>-FliZ
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    <td>FliZ</td>
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<br/>-MotB
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    <td>45&deg;C</td>
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<br/>-EstA
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    <td>130</td>
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<br/>-LytB
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  </tr>
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<br/>The size will be around 100-150 bp.
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  <tr>
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    <td>MotB</td>
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    <td>50&deg;C</td>
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    <td>100-150</td>
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  </tr>
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The following protocol is used for the PCR of EstA, MotB and LytB:
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  <tr>
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<br/>98°C, 98°C, 50°C, 72°C, 72°C, 4°C
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    <td>EstA</td>
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<br/>0:30  0:10  0:25  0:05  10:00 forever
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    <td>50&deg;C</td>
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    <td>100-150</td>
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  </tr>
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The following protocol is used for the PCR of FliZ:
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  <tr>
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<br/>98°C, 98°C, 45°C, 72°C, 72°C, 4°C
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    <td>LytB</td>
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<br/>0:30  0:10  0:25  0:04  10:00 forever
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    <td>50&deg;C</td>
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    <td>100-150</td>
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  </tr>
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Run a 1.5% agarose gel for 13 minutes. Examination of the gel revealed that all expected products are there. The picture will follow.
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</table>
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<br>The PCR product is loaded on <a href="https://2013.igem.org/Team:Groningen/protocols/GelElectrophoresis"><FONT COLOR="black"><b>gel</b></FONT></a> to examine if the PCR reaction succeeded.
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<br>picture
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<p>
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<h2>Claudio and Inne</h2>
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Further discussion about the available options to create a heat-attracted bacteria.
      
      
</div>
</div>

Latest revision as of 16:17, 28 July 2013

Mirjam

Did a genomic DNA extraction of B. subtilis strain 168. Two tubes are obtained from this with a concentration of 66ng/µl and 67ng/µl.

The primers for our main subject, silk secretion, arrived. The primers are diluted to a concentration of 10mM.
To run a PCR, dNTPs are necessary in a concentration of 10mM, so this dilution is also made.

A PCR reaction mix is made for the four different signal sequences.

signal sequence Tm-5 size
FliZ 45°C 130
MotB 50°C 100-150
EstA 50°C 100-150
LytB 50°C 100-150

The PCR product is loaded on gel to examine if the PCR reaction succeeded.
picture

Claudio and Inne

Further discussion about the available options to create a heat-attracted bacteria.

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