Team:Groningen/protocols/Motility assay

From 2013.igem.org

(Difference between revisions)
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<br>The strains were grown overnight at 37°C on LB broth.
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<h2>Motility assay</h2>
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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.  
+
Ad 13 ml of the motility agar (0.4% or 0.7%) per plate.
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<br> Sgg is 2x SG supplemented with 1% [wt/vol] glycerol.
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<p>Inoculate the plates at the center with the Strain of interest (strain A).  
 +
<p>For every plate inoculated with Strain A inoculate a plate with Wild type (WT).
 +
<p>inoculations is done with 10 ul of liquid culture with an OD<sub>600</sub> of 0.4.
 +
<p>Let the colonies grow over 16 hours.
 +
<p>After the 16 hour growth period the measure the spread of the colony every 2 hours.
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Motility Agar 0.7% (100 ml)  
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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">
<table id="normal">
   <tr>
   <tr>
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   <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 ALIGN=CENTER>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>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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+
-
  <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>
     <td></td>
   </tr>
   </tr>
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   <tr>
   <tr>
     <td>agar</td>
     <td>agar</td>
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     <td ALIGN=CENTER>1.5 g</td>
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     <td ALIGN=CENTER>0.7 g</td>
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    <td> </td>
+
-
  </tr>
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+
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<tr>
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     <td></td>
     <td></td>
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    <td ALIGN=CENTER></td>
 
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    <td> add everything together and autoclave</td>
 
   </tr>
   </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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<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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<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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Motility Agar 0.4% (100 ml)  
<table id="normal">
<table id="normal">
   <tr>
   <tr>
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   <tr>
   <tr>
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     <td>LB/ 1.5% agar</td>
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     <td>LB broth </td>
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     <td ALIGN=CENTER>100 ml</td>
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     <td ALIGN=CENTER>2 g</td>
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    <td>autoclave</td>
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  </tr>
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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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<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>
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    <th>compound</th>
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    <th>amount</th>
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    <th>treatment</th>
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  </tr>
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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>
     <td></td>
   </tr>
   </tr>
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   <tr>
   <tr>
     <td>agar</td>
     <td>agar</td>
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     <td ALIGN=CENTER>1.5 g</td>
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     <td ALIGN=CENTER>0.4 g</td>
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    <td> </td>
+
-
  </tr>
+
-
 
+
-
<tr>
+
     <td></td>
     <td></td>
-
    <td ALIGN=CENTER></td>
 
-
    <td>add everything together and autoclave</td>
 
   </tr>
   </tr>
-
 
-
<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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<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>
 
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  </tr>
 
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<tr>
 
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    <td>FeCl<sub>3</sub>  50 &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>ZnCl<sub>2</sub> 1 &micro;M</td>
 
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    <td ALIGN=CENTER>0.1 mL</td>
 
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    <td> </td>
 
-
  </tr>
 
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-
<tr>
 
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    <td>thiamine 2 &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>0.5% glycerol </td>
 
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    <td ALIGN=CENTER>0.57 mL</td>
 
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    <td> </td>
 
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  </tr>
 
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<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>
 
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<tr>
 
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    <td>tryptophan </td>
 
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    <td ALIGN=CENTER>1 mL</td>
 
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    <td> </td>
 
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  </tr>
 
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</table>
 
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<p>
 
-
 
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</p>
 

Revision as of 10:52, 4 October 2013

Motility assay

Ad 13 ml of the motility agar (0.4% or 0.7%) per plate.

Inoculate the plates at the center with the Strain of interest (strain A).

For every plate inoculated with Strain A inoculate a plate with Wild type (WT).

inoculations is done with 10 ul of liquid culture with an OD600 of 0.4.

Let the colonies grow over 16 hours.

After the 16 hour growth period the measure the spread of the colony every 2 hours. Motility Agar 0.7% (100 ml)

Motility Agar 0.4% (100 ml)
compound amount treatment
LB broth 2 g
agar 0.7 g
compound amount treatment
LB broth 2 g
agar 0.4 g