Team:Groningen/Labwork/26 June 2013

From 2013.igem.org

(Difference between revisions)
 
(4 intermediate revisions not shown)
Line 24: Line 24:
      
      
<h2>Mirjam</h2>
<h2>Mirjam</h2>
-
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
+
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.
-
Diluting the primers (to 100 mM and 10 mM)
+
<p>The primers for our main subject, silk secretion, arrived. The primers are diluted to a concentration of 10mM.
-
<br/>Diluting the dNTPs to a concentration of 10 mM.  
+
<br>To run a PCR, dNTPs are necessary in a concentration of 10mM, so this dilution is also made.
-
A PCR reaction mix is made containing the following compounds:
+
<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.
-
<br/>5x Buffer HF  1x
+
-
<br/>dNTPs          200 µM each
+
-
<br/>primer F      1 µM
+
-
<br/>primer R      1 µM
+
-
<br/>temp DNA      6.7 ng
+
-
<br/>phusion pol.  0.02 U/µl
+
-
<br/>MQ water is added to get the wanted volume.
+
 +
<br>
 +
<table border="1">
 +
  <tr>
 +
    <th>signal sequence</th>
 +
    <th>T<sub>m</sub>-5</th>
 +
    <th>size</th>
 +
  </tr>
-
Run a PCR for the four different signal sequences.
+
  <tr>
-
<br/>-FliZ
+
    <td>FliZ</td>
-
<br/>-MotB
+
    <td>45&deg;C</td>
-
<br/>-EstA
+
    <td>130</td>
-
<br/>-LytB
+
  </tr>
-
<br/>The size will be around 100-150 bp.
+
 +
  <tr>
 +
    <td>MotB</td>
 +
    <td>50&deg;C</td>
 +
    <td>100-150</td>
 +
  </tr>
-
The following protocol is used for the PCR of EstA, MotB and LytB:
+
  <tr>
-
<br/>98°C, 98°C, 50°C, 72°C, 72°C, 4°C
+
    <td>EstA</td>
-
<br/>0:30  0:10  0:25  0:05  10:00 forever
+
    <td>50&deg;C</td>
 +
    <td>100-150</td>
 +
  </tr>
-
The following protocol is used for the PCR of FliZ:
+
  <tr>
-
<br/>98°C, 98°C, 45°C, 72°C, 72°C, 4°C
+
    <td>LytB</td>
-
<br/>0:30  0:10  0:25  0:04  10:00 forever
+
    <td>50&deg;C</td>
 +
    <td>100-150</td>
 +
  </tr>
-
Run a 1.5% agarose gel for 13 minutes. Examination of the gel revealed that all expected products are there. The picture will follow.
+
</table>
-
<h2>Claudio and Inne</h2>
+
<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.
 +
<br>picture
-
<br>Further discussion about the available options to create a neat-attracted bacteria a discussed.
+
<p>
 +
<h2>Claudio and Inne</h2>
 +
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.

Retrieved from "http://2013.igem.org/Team:Groningen/Labwork/26_June_2013"