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>
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<h1><a href="https://2014.igem.org/Team:Hannover/Protocols">Protocols</a> / SDS-PAGE</h1>
<table colspan="2"><tr><td><h4>Material:</h4></td>
<table colspan="2"><tr><td><h4>Material:</h4></td>
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<tr><td>12 % Running gel</td><td>2.1 ml  H<sub>2</sub>O,<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  H<sub>2</sub>O,<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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<tr><td>12 % Running gel</td><td>2.1 ml  H<sub>2</sub>O,<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  H<sub>2</sub>O,<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>
<h2>Protocol</h2>
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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 H<sub>2</sub>O 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 H<sub>2</sub>O 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 to 1:20 h at 150 V.</p>
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<p class="text">The SDS polyacrylamide gels are prepared in the so-called PerfectBlue™ Twin Double Gel System (PEQLAB Biotechnologie GmbH, Erlangen). After ensuring that the equipment is waterproof, the 12 % running gel is mixed and filled into the chamber. Pipetting 1 ml of H<sub>2</sub>O on top of the running gel, prevents coves between the entire running and stacking gel. The comb is stuck into the chamber to maintain the right gel depth. After polymerization, the remaining H<sub>2</sub>O is removed and the 12 % stacking gel (Table 1) is filled on top. To create sample pockets a comb is inserted centrally. If the stacking gel is also polymerized, 1 x running buffer (Table 1)is used to run the Double Gel System via the SDS gel. After loading the generated pockets with the samples, the gel is run for 1:10 to 1:20 h at 150 V.</p>
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Latest revision as of 23:37, 17 October 2014

Protocols / SDS-PAGE

Material:
12 % Running gel2.1 ml H2O,
2.5 ml acrylamide 4K 29:1 mix (40 %),
1.6 ml 1.5 M Tris-HCl; pH 8.8,
62.5 μl 10 % SDS,
62.5 μl 10 % APS,
2.5 μl TEMED
12 % Stacking gel2.7 ml H2O,
680 μl acrylamide 4K 29:1 mix (40 %),
540 μl 1 M Tris-HCl; pH 6.8,
40 μl 10 % SDS,
30 μl 10 % APS,
8 μl TEMED
Electrophoresis system
Running buffer250 mM Tris-HCl; pH 8.3,
1.92 mM glycine,
0.1 % (v/v) SDS


Protocol

The SDS polyacrylamide gels are prepared in the so-called PerfectBlue™ Twin Double Gel System (PEQLAB Biotechnologie GmbH, Erlangen). After ensuring that the equipment is waterproof, the 12 % running gel is mixed and filled into the chamber. Pipetting 1 ml of H2O on top of the running gel, prevents coves between the entire running and stacking gel. The comb is stuck into the chamber to maintain the right gel depth. After polymerization, the remaining H2O is removed and the 12 % stacking gel (Table 1) is filled on top. To create sample pockets a comb is inserted centrally. If the stacking gel is also polymerized, 1 x running buffer (Table 1)is used to run the Double Gel System via the SDS gel. After loading the generated pockets with the samples, the gel is run for 1:10 to 1:20 h at 150 V.

Polyacrylamide is a strong toxic agent for neurons. Be careful please!





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