Team:Hannover/Protocols/SDS PAGE

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<h1><a href="https://2014.igem.org/Team:Hannover/Protocols" target="_blank">Protocols</a> / SDS-PAGE</h1>
<table colspan="2"><tr><td><h4>Material:</h4></td>
<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 %)<span id='a1'</span>,<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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<h2>Protocol</h2>
<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'>Polyacrylamide is a strong toxic agent for neurons. Be careful please!</div>
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<p id="Fusszeile"><br><br><br><br><img src="https://static.igem.org/mediawiki/2014/6/6a/Hannover_20141010Fusszeile2.jpg" width="980px" style="float:right" /></p>
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