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Team:Hannover/Protocols/SDS PAGE
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| - | <h1><a href="https://2014.igem.org/Team:Hannover/Protocols | + | <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> | ||
| - | <tr><td>12 % Running gel</td><td>2.1 ml | + | <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> |
<br><br> | <br><br> | ||
<h2>Protocol</h2> | <h2>Protocol</h2> | ||
| - | <p class="text"> | + | <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 gel | 2.1 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 gel | 2.7 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 buffer | 250 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!
