Team:Hannover/Protocols/Detection/Precipitation

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<h1><a href="https://2014.igem.org/Team:Hannover/Protocols" target="_blank">Protocols</a> / Precipitating Bacteria for MS Analysis</h1>
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<div ref="kathi3" class="annotation" style="top: 395.333px; left: 780px; visibility: visible;">If you have to deal with heavy metals, choose nitril gloves and change them once you came in contact with them!</a>. </div>
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<h1><a href="https://2014.igem.org/Team:Hannover/Protocols">Protocols</a> / Precipitating Bacteria for MS or ICP-OES Analysis</h1>
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<table colspan="2"><tr><td><h4>Material:</h4></td>
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<table colspan="2" width="250"><tr><td><h4>Material:</h4></td>
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<tr><td>12 % Running gel</td><td>2.1 ml  H2O,<br>2.5 ml  acrylamide 4K 29:1 mix  (40 %),<br> 1.6 ml 1.5 M Tris-HCl pH 8.8,<br> 62.5 μl 10 % SDS,<br> 62.5 μl 10 % APS,<br> 2.5 μl TEMED</td></tr><tr><td></td><td></td></tr><tr><td>12 % Stacking gel</td><td>2.7 ml  H2O,<br>680 μl  acrylamide 4K  29:1 mix  (40 %),<br>540 μl 1 M Tris-HCl pH 6.8,<br>40 μl 10 % SDS,<br>30 μl 10 % APS,<br>8 μl TEMED</td></tr><tr><td>Electrophoresis system</td><td></td></tr><tr><td></td><td></td></tr><tr><td>Running buffer</td><td>250 mM Tris-HCl pH 8.3,<br>1.92 mM glycine,<br> 0,1 % (v/v) SDS</td></tr></table>
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<tr><td>gloves</td><td></td></tr></tr><span id='a2'</span>
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<tr><td>incubator</td><td></td></tr>
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<tr><td>Isoosmotic buffer</td><td>Prepare 2 l,<br> 50 mM Tris-HCl,<br> 10 mM EDTA; pH 8.0</td></tr></table>
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<br><br><span id='a1'></span>  
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<h2>Protocol</h2>
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<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>
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<p><h4>Protocol:</h4></p>
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<p class="text">After <i>E. coli</i>SDS polyacrylamide gels were prepared in the so-called PerfectBlue™ Twin Double Gel System (PEQLAB Biotechnologie GMBH, Erlangen). After ensuring the aperture was waterproof, the 12 % running gel was mixed and filled into the chamber. Pipetting 1 ml of H2O on top of the running gel prevented coves between the entire running and stacking gel. The comb was stuck into the chamber to maintain the right gel depth. After polymerization, the remaining H2O was removed and the 12 % stacking gel (Table 1) followed. To create sample pockets a comb was inserted centrally. If the stacking gel was also polymerized, 1 x running buffer (Table 1) was used to run the Double Gel System via the SDS gel. After loading the generated pockets with the samples, the gel was run for 1:10 1:20 h at 150 V.</p>
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<div class='annotation' ref='a1'>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!</div>
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<div class="annotation" ref='a2'>If you have to deal with heavy metals, choose thicker nitril gloves. Be aware that washing by warm water increases the gloves´permeability!</a>.</div></div>
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Latest revision as of 23:37, 17 October 2014

Protocols / Precipitating Bacteria for MS or ICP-OES Analysis

Material:
gloves
incubator
Isoosmotic bufferPrepare 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!.





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