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Team:Hannover/Protocols/SDS PAGE
From 2014.igem.org
Protocols / SDS-PAGE
Material: |
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| 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
After E. coli 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 to 1:20 h at 150 V.
