Team:Groningen/protocols/Motility assay

From 2013.igem.org

(Difference between revisions)
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<div class="mainContent">
<div class="mainContent">
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<h2>Motility assay</h2>
 
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Ad 13 ml of the motility agar (0.4% or 0.7%) per plate.
 
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<p>Inoculate the plates at the center with the Strain of interest (strain A).
 
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<p>For every plate inoculated with Strain A inoculate a plate with Wild type (WT).
 
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<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.
 
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<p>After the 16 hour growth period the measure the spread of the colony every 2 hours.
 
-
Motility Agar 0.7% (100 ml)  
+
<br>The strains were grown overnight at 37°C on LB broth.  
 +
<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.
 +
<br> Sgg is 2x SG supplemented with 1% [wt/vol] glycerol.
 +
 
 +
 
 +
 
 +
<h2>Media for macrocolony formation</h2>
 +
 
 +
<br><H5>2x SG medium (100 mL)</H5>
<table id="normal">
<table id="normal">
   <tr>
   <tr>
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   <tr>
   <tr>
-
     <td>LB broth </td>
+
     <td>Nutrient broth (Difco)</td>
-
     <td ALIGN=CENTER>2 g</td>
+
     <td ALIGN=CENTER>1.6 g</td>
 +
    <td></td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>KCl</td>
 +
    <td ALIGN=CENTER>0.2 g</td>
 +
    <td></td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>MgSO<sub>4</sub>.7H<sub>2</sub>O </td>
 +
    <td ALIGN=CENTER>0.05 g</td>
     <td></td>
     <td></td>
   </tr>
   </tr>
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   <tr>
   <tr>
     <td>agar</td>
     <td>agar</td>
-
     <td ALIGN=CENTER>0.7 g</td>
+
     <td ALIGN=CENTER>1.5 g</td>
 +
    <td> </td>
 +
  </tr>
 +
 
 +
<tr>
     <td></td>
     <td></td>
 +
    <td ALIGN=CENTER></td>
 +
    <td> add everything together and autoclave</td>
   </tr>
   </tr>
 +
 +
<tr>
 +
    <td>Ca(NO<sub>3</sub>)<sub>2</sub>.4H<sub>2</sub>O  1 mM</td>
 +
    <td ALIGN=CENTER>0.1 mL</td>
 +
    <td> </td>
 +
  </tr>
 +
 +
<tr>
 +
    <td>MnCl<sub>2</sub>.4H<sub>2</sub>O 0.1 mM</td>
 +
    <td ALIGN=CENTER>0.1 mL</td>
 +
    <td> </td>
 +
  </tr>
 +
 +
<tr>
 +
    <td>FeSO<sub>4</sub>  1 &micro;M</td>
 +
    <td ALIGN=CENTER>0.1 mL</td>
 +
    <td> </td>
 +
  </tr>
 +
 +
<tr>
 +
    <td>0.1% glucose </td>
 +
    <td ALIGN=CENTER>0.5 mL</td>
 +
    <td> </td>
 +
  </tr>
 +
</table>
-
Motility Agar 0.4% (100 ml)  
+
<br><H5>LB/Mn/glucose medium (100 mL)</H5>
<table id="normal">
<table id="normal">
   <tr>
   <tr>
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   <tr>
   <tr>
-
     <td>LB broth </td>
+
     <td>LB/ 1.5% agar</td>
-
     <td ALIGN=CENTER>2 g</td>
+
     <td ALIGN=CENTER>100 ml</td>
 +
    <td>autoclave</td>
 +
  </tr>
 +
 
 +
<tr>
 +
    <td>MnCl<sub>2</sub>.4H<sub>2</sub>O  0.1 mM</td>
 +
    <td ALIGN=CENTER>0.1mL</td>
 +
    <td> </td>
 +
  </tr>
 +
 
 +
<tr>
 +
    <td>0.1% glucose </td>
 +
    <td ALIGN=CENTER>0.5 mL</td>
 +
    <td> </td>
 +
  </tr>
 +
</table>
 +
 
 +
 
 +
 
 +
 
 +
<br><H5> MSgg medium (100 mL)</H5>
 +
<table id="normal">
 +
  <tr>
 +
    <th>compound</th>
 +
    <th>amount</th>
 +
    <th>treatment</th>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>KH<sub>2</sub>PO<sub>4</sub></td>
 +
    <td ALIGN=CENTER>0.026 g</td>
 +
    <td></td>
 +
  </tr>
 +
<tr>
 +
    <td>K<sub>2</sub>HPO<sub>4</sub></td>
 +
    <td ALIGN=CENTER>0.061 g</td>
 +
    <td></td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>MOPS 100 mM</td>
 +
    <td ALIGN=CENTER>2.09 g</td>
 +
    <td></td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>MgCl<sub>2</sub>.6H<sub>2</sub>O  2 mM</td>
 +
    <td ALIGN=CENTER>0.04 g</td>
     <td></td>
     <td></td>
   </tr>
   </tr>
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   <tr>
   <tr>
     <td>agar</td>
     <td>agar</td>
-
     <td ALIGN=CENTER>0.4 g</td>
+
     <td ALIGN=CENTER>1.5 g</td>
 +
    <td> </td>
 +
  </tr>
 +
 
 +
<tr>
     <td></td>
     <td></td>
 +
    <td ALIGN=CENTER></td>
 +
    <td>add everything together and autoclave</td>
 +
  </tr>
 +
 +
<tr>
 +
    <td>CaCl<sub>2</sub>  700 mM</td>
 +
    <td ALIGN=CENTER>0.1 mL</td>
 +
    <td> </td>
 +
  </tr>
 +
 +
<tr>
 +
    <td>MnCl<sub>2</sub> 50 &micro;M</td>
 +
    <td ALIGN=CENTER>50 &micro;L</td>
 +
    <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>
 +
    <td ALIGN=CENTER>0.1 mL</td>
 +
    <td> </td>
 +
  </tr>
 +
 +
<tr>
 +
    <td>thiamine 2 &micro;M</td>
 +
    <td ALIGN=CENTER>0.1 mL</td>
 +
    <td> </td>
 +
  </tr>
 +
 +
<tr>
 +
    <td>0.5% glycerol </td>
 +
    <td ALIGN=CENTER>0.57 mL</td>
 +
    <td> </td>
 +
  </tr>
 +
 +
<tr>
 +
    <td>0.5% glutamate </td>
 +
    <td ALIGN=CENTER>10 mL</td>
 +
    <td> </td>
 +
  </tr>
 +
 +
<tr>
 +
    <td>tryptophan </td>
 +
    <td ALIGN=CENTER>1 mL</td>
 +
    <td> </td>
   </tr>
   </tr>
 +
</table>
 +
<p>
 +
</p>

Revision as of 10:52, 4 October 2013


The strains were grown overnight at 37°C on LB broth.
Next, 1 ml of liquid culture was used to inoculate every 10 ml 2x Sgg medium. this in turn was incubated at 27°C for 5 days.
Sgg is 2x SG supplemented with 1% [wt/vol] glycerol.

Media for macrocolony formation


2x SG medium (100 mL)
compound amount treatment
Nutrient broth (Difco) 1.6 g
KCl 0.2 g
MgSO4.7H2O 0.05 g
agar 1.5 g
add everything together and autoclave
Ca(NO3)2.4H2O 1 mM 0.1 mL
MnCl2.4H2O 0.1 mM 0.1 mL
FeSO4 1 µM 0.1 mL
0.1% glucose 0.5 mL

LB/Mn/glucose medium (100 mL)
compound amount treatment
LB/ 1.5% agar 100 ml autoclave
MnCl2.4H2O 0.1 mM 0.1mL
0.1% glucose 0.5 mL

MSgg medium (100 mL)
compound amount treatment
KH2PO4 0.026 g
K2HPO4 0.061 g
MOPS 100 mM 2.09 g
MgCl2.6H2O 2 mM 0.04 g
agar 1.5 g
add everything together and autoclave
CaCl2 700 mM 0.1 mL
MnCl2 50 µM 50 µL
FeCl3 50 µM 0.1 mL
ZnCl2 1 µM 0.1 mL
thiamine 2 µM 0.1 mL
0.5% glycerol 0.57 mL
0.5% glutamate 10 mL
tryptophan 1 mL