Team:Groningen/protocols/Motility assay

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<h2>Motility assay</h2>
<h2>Motility assay</h2>
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<p>A motility assay is done for <i>Bacillus subtilus</i> of different knockout strains as wel as a wild type strain.  
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Ad 13 ml of the motility agar (0.4% or 0.7%) per plate.
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<p>This is to determine how different the knockouts move compared to the wild type <i>B. subtilus</i>.
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<p>Inoculate the plates at the center with the Strain of interest (strain A).
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<p>The results will be used to determine if the temperature controlled strain works as desired, moving more as a CheY knockout in warm environments and more as a wild type in cold environments.
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<p>For every plate inoculated with Strain A inoculate a plate with Wild type (WT).  
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<p>The design of the motility assay is a simple one. LB agar Plates are made with the normal amount of LB-broth as nutrients but with a reduced amount of agar.
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<p>inoculations is done with 10 ul of liquid culture with an OD<sub>600</sub> of 0.4.
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<p>Different concentrations are used to find out the optimal amount of agar to show the motility.
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<p>Let the colonies grow over 16 hours.
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Low concentrations are needed to allow movement through the medium but too low and the observed movement can be caused by dispersal and turbulence during movement.  
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<p>After the 16 hour growth period the measure the spread of the colony every 2 hours.
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The concentrations that are used are, 0.4% agar and 0.7% agar. The plates contained 13 ml agar.
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<p>The plates are inoculated in the center with the strain that is to be tested. This is done with 10 ul of liquid culture at an OD of 0.4. The inoculation was done by sticking the pipet in to the agar, injecting the sample in to it.
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Motility Agar 0.7% (100 ml)
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<p>For each strain 9 plates of each agar concentration is made. Three of the plates are placed in a 37 C stove and three placed in a 30 C stove and three placed in a 27 C stove to grow.
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<table id="normal">
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<p>The colonys are left to grow for 16 hours. Then how the colonies spread on the plates are observed every hour. The bacteria that are more motile should spread out over the agar creating a clouwdy look while the non- motile bacteria should stay in the center, close together.
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  <tr>
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<p>How quickly the bacteria spread from the center to the edge can be used as an indicatore of how fast they move.
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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>LB broth </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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  <tr>
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    <td>agar</td>
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    <td ALIGN=CENTER>0.7 g</td>
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    <td></td>
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  </tr>
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 +
 
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Motility Agar 0.4% (100 ml)
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<table id="normal">
 +
  <tr>
 +
    <th>compound</th>
 +
    <th>amount</th>
 +
    <th>treatment</th>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>LB broth </td>
 +
    <td ALIGN=CENTER>2 g</td>
 +
    <td></td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>agar</td>
 +
    <td ALIGN=CENTER>0.4 g</td>
 +
    <td></td>
 +
  </tr>
 +
 
 +
 
 +
 
 +
 
</div>
</div>

Revision as of 10:44, 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

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