Team:Groningen/protocols/Motility assay

From 2013.igem.org

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<br>The strains were grown overnight at 37°C on LB broth.  
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<h1>Motility assay</h1>
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<br>Next, 1 ml of liquid culture was used to inoculate every 10 ml 2x Sgg medium. this in turn was incubated at 27&deg;C for 5 days.  
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Add 13 ml of the motility agar (0.4%) in every petri dish.
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<br> Sgg is 2x SG supplemented with 1% [wt/vol] glycerol.
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<br>Inoculate the plates with the strain of interest (10 &micro;l of overnight culture with an OD<sub>600</sub> of 0.4).
 +
<br>The spot is dried for half an hour, after which the plates are inoculated at different temperatures (25&deg;C and 37&deg;C) for 16 hours.
 +
<br>After 16 hours of growth, the diameter of the colony is measured.  
 +
<p>
 +
<b>Motility Agar 0.4% (100 ml) </b>
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</p>
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<table id="normal" width="60%">
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<h2>Media for macrocolony formation</h2>
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<br><H5>2x SG medium (100 mL)</H5>
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<table id="normal">
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   <tr>
   <tr>
     <th>compound</th>
     <th>compound</th>
     <th>amount</th>
     <th>amount</th>
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    <th>treatment</th>
 
   </tr>
   </tr>
   <tr>
   <tr>
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     <td>Nutrient broth (Difco)</td>
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     <td>LB broth </td>
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     <td ALIGN=CENTER>1.6 g</td>
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     <td>2 g</td>
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    <td></td>
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  </tr>
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  <tr>
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    <td>KCl</td>
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    <td ALIGN=CENTER>0.2 g</td>
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    <td></td>
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  </tr>
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+
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  <tr>
+
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    <td>MgSO<sub>4</sub>.7H<sub>2</sub>O </td>
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    <td ALIGN=CENTER>0.05 g</td>
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    <td></td>
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   </tr>
   </tr>
   <tr>
   <tr>
     <td>agar</td>
     <td>agar</td>
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     <td ALIGN=CENTER>1.5 g</td>
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     <td>0.4 g</td>
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    <td> </td>
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   </tr>
   </tr>
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</table>
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<tr>
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    <td></td>
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    <td ALIGN=CENTER></td>
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    <td> add everything together and autoclave</td>
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  </tr>
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<tr>
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    <td>Ca(NO<sub>3</sub>)<sub>2</sub>.4H<sub>2</sub>O  1 mM</td>
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    <td ALIGN=CENTER>0.1 mL</td>
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    <td> </td>
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  </tr>
+
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+
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<tr>
+
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    <td>MnCl<sub>2</sub>.4H<sub>2</sub>O 0.1 mM</td>
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    <td ALIGN=CENTER>0.1 mL</td>
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    <td> </td>
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  </tr>
+
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+
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<tr>
+
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    <td>FeSO<sub>4</sub>  1 &micro;M</td>
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    <td ALIGN=CENTER>0.1 mL</td>
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    <td> </td>
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-
  </tr>
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<tr>
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    <td>0.1% glucose </td>
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    <td ALIGN=CENTER>0.5 mL</td>
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    <td> </td>
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  </tr>
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</table>  
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<br><H5>LB/Mn/glucose medium (100 mL)</H5>
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<table id="normal">
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-
  <tr>
+
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    <th>compound</th>
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    <th>amount</th>
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    <th>treatment</th>
+
-
  </tr>
+
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+
-
  <tr>
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    <td>LB/ 1.5% agar</td>
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    <td ALIGN=CENTER>100 ml</td>
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    <td>autoclave</td>
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  </tr>
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+
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<tr>
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    <td>MnCl<sub>2</sub>.4H<sub>2</sub>O  0.1 mM</td>
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    <td ALIGN=CENTER>0.1mL</td>
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    <td> </td>
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  </tr>
+
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+
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<tr>
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    <td>0.1% glucose </td>
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    <td ALIGN=CENTER>0.5 mL</td>
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    <td> </td>
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  </tr>
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</table>
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+
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+
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+
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<br><H5> MSgg medium (100 mL)</H5>
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<table id="normal">
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-
  <tr>
+
-
    <th>compound</th>
+
-
    <th>amount</th>
+
-
    <th>treatment</th>
+
-
  </tr>
+
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+
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  <tr>
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    <td>KH<sub>2</sub>PO<sub>4</sub></td>
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    <td ALIGN=CENTER>0.026 g</td>
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    <td></td>
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  </tr>
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<tr>
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    <td>K<sub>2</sub>HPO<sub>4</sub></td>
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    <td ALIGN=CENTER>0.061 g</td>
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    <td></td>
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  </tr>
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  <tr>
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    <td>MOPS 100 mM</td>
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    <td ALIGN=CENTER>2.09 g</td>
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    <td></td>
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  </tr>
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  <tr>
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    <td>MgCl<sub>2</sub>.6H<sub>2</sub>O  2 mM</td>
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    <td ALIGN=CENTER>0.04 g</td>
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    <td></td>
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  </tr>
+
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+
-
  <tr>
+
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    <td>agar</td>
+
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    <td ALIGN=CENTER>1.5 g</td>
+
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    <td> </td>
+
-
  </tr>
+
-
 
+
-
<tr>
+
-
    <td></td>
+
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    <td ALIGN=CENTER></td>
+
-
    <td>add everything together and autoclave</td>
+
-
  </tr>
+
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+
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<tr>
+
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    <td>CaCl<sub>2</sub>  700 mM</td>
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    <td ALIGN=CENTER>0.1 mL</td>
+
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    <td> </td>
+
-
  </tr>
+
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+
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<tr>
+
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    <td>MnCl<sub>2</sub> 50 &micro;M</td>
+
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    <td ALIGN=CENTER>50 &micro;L</td>
+
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    <td> </td>
+
-
  </tr>
+
-
 
+
-
<tr>
+
-
    <td>FeCl<sub>3</sub>  50 &micro;M</td>
+
-
    <td ALIGN=CENTER>0.1 mL</td>
+
-
    <td> </td>
+
-
  </tr>
+
-
 
+
-
<tr>
+
-
    <td>ZnCl<sub>2</sub> 1 &micro;M</td>
+
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    <td ALIGN=CENTER>0.1 mL</td>
+
-
    <td> </td>
+
-
  </tr>
+
-
 
+
-
<tr>
+
-
    <td>thiamine 2 &micro;M</td>
+
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    <td ALIGN=CENTER>0.1 mL</td>
+
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    <td> </td>
+
-
  </tr>
+
-
 
+
-
<tr>
+
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    <td>0.5% glycerol </td>
+
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    <td ALIGN=CENTER>0.57 mL</td>
+
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    <td> </td>
+
-
  </tr>
+
-
 
+
-
<tr>
+
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    <td>0.5% glutamate </td>
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    <td ALIGN=CENTER>10 mL</td>
+
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    <td> </td>
+
-
  </tr>
+
-
 
+
-
<tr>
+
-
    <td>tryptophan </td>
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    <td ALIGN=CENTER>1 mL</td>
+
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    <td> </td>
+
-
  </tr>
+
-
</table>
+
-
<p>
+
-
 
+
-
</p>
+
-
 
+

Latest revision as of 00:22, 5 October 2013

Motility assay

Add 13 ml of the motility agar (0.4%) in every petri dish.
Inoculate the plates with the strain of interest (10 µl of overnight culture with an OD600 of 0.4).
The spot is dried for half an hour, after which the plates are inoculated at different temperatures (25°C and 37°C) for 16 hours.
After 16 hours of growth, the diameter of the colony is measured.

Motility Agar 0.4% (100 ml)

compound amount
LB broth 2 g
agar 0.4 g