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Team:Hannover/Protocols/Detection/Precipitation
From 2014.igem.org
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<tr><td>gloves</td><td></td></tr></tr><span id='a2'</span> | <tr><td>gloves</td><td></td></tr></tr><span id='a2'</span> | ||
<tr><td>incubator</td><td></td></tr> | <tr><td>incubator</td><td></td></tr> | ||
| - | <tr><td> | + | <tr><td>Isoosmotic buffer</td><td>Prepare 2 l,<br> 50 mM Tris-HCl,<br> 10 mM EDTA; pH 8.0</td></tr></table> |
<br><br><span id='a1'></span> | <br><br><span id='a1'></span> | ||
<h2>Protocol</h2> | <h2>Protocol</h2> | ||
| - | <p class="text">After induction of heterologous T4MBP expression, | + | <p class="text">After induction of heterologous T4MBP expression, bacterial cell-cultures were centrifuged for 15 min at 4500 x g and 4 °C. While the supernatant was decanted, the precipitated bacteria were dissolved in 20 ml isoosmotic buffer. According to the harm of heavy metals, all medium supernatants were stored and had to be picked up by professional disposal service. Starting with the dissolving the precipitate, the washing is repeated four more times. Due to the heavy metals, the supernatants obtained here had to be stored also. As the last step, precipitated bacteria were dissolved in 1 ml H<sub>2</sub>O and dried for 2 d at 60 °C. For latter MS, the reaction tube´s dry-weight before and after loading the aqueous bacteria solution had to be determined.</p> |
</div> | </div> | ||
Revision as of 19:25, 17 October 2014
Protocols / Precipitating Bacteria for MS Analysis
Material: |
|
| gloves | |
| incubator | |
| Isoosmotic buffer | Prepare 2 l, 50 mM Tris-HCl, 10 mM EDTA; pH 8.0 |
Protocol
After induction of heterologous T4MBP expression, bacterial cell-cultures were centrifuged for 15 min at 4500 x g and 4 °C. While the supernatant was decanted, the precipitated bacteria were dissolved in 20 ml isoosmotic buffer. According to the harm of heavy metals, all medium supernatants were stored and had to be picked up by professional disposal service. Starting with the dissolving the precipitate, the washing is repeated four more times. Due to the heavy metals, the supernatants obtained here had to be stored also. As the last step, precipitated bacteria were dissolved in 1 ml H2O and dried for 2 d at 60 °C. For latter MS, the reaction tube´s dry-weight before and after loading the aqueous bacteria solution had to be determined.
Some bacteria grown in high heavy metal concentrations will not precipitate by centrifuging. Thus it will be very hard to remove the supernatant. Don’t worry if some of the precipitated fraction unfortunately falls out during decanting. Then, make sure that you treat all samples in the same manner!
If you have to deal with heavy metals, choose thicker nitril gloves. Be aware that washing by warm water increases the gloves´permeability!.
