Team:Groningen/protocols/Motility assay
From 2013.igem.org
(Difference between revisions)
Line 22: | Line 22: | ||
<div class="mainContent"> | <div class="mainContent"> | ||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | + | <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°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> | ||
Line 39: | Line 40: | ||
<tr> | <tr> | ||
- | <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> | ||
Line 46: | Line 59: | ||
<tr> | <tr> | ||
<td>agar</td> | <td>agar</td> | ||
- | <td ALIGN=CENTER> | + | <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 µM</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> | ||
- | + | <br><H5>LB/Mn/glucose medium (100 mL)</H5> | |
<table id="normal"> | <table id="normal"> | ||
<tr> | <tr> | ||
Line 61: | Line 105: | ||
<tr> | <tr> | ||
- | <td>LB | + | <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> | ||
Line 68: | Line 159: | ||
<tr> | <tr> | ||
<td>agar</td> | <td>agar</td> | ||
- | <td ALIGN=CENTER> | + | <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 µM</td> | ||
+ | <td ALIGN=CENTER>50 µL</td> | ||
+ | <td> </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>FeCl<sub>3</sub> 50 µM</td> | ||
+ | <td ALIGN=CENTER>0.1 mL</td> | ||
+ | <td> </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>ZnCl<sub>2</sub> 1 µM</td> | ||
+ | <td ALIGN=CENTER>0.1 mL</td> | ||
+ | <td> </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>thiamine 2 µM</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 |