ESO wikiadmin: Created page with "{| class="wikitable" |+ !Materials ! |- |Separating Gel (12,5%): |3.1 mL Polyacrylamide (40%); 4.3 mL dH₂O; 2.5 mL Tris (1.5 M; pH 8.8) ''Degas!'' 50 μL Sodium dodecyl sulfate (SDS; 20%); 30 μL Ammonium Persulfate (APS; 10%); 15 μL TEMED |- |Stacking Gel (5%): |1.25 mL Polyacrylamide (40%); 6.15 mL dH₂O; 2.5 mL Tris (0,5 M; pH 6,8) ''Degas!'' 50 μL Sodium dodecyl sulfate (SDS; 20%); 30 μL Ammonium Persulfate (APS; 10%); 15 μL TEMED |- |Marker..."

2025-02-27T13:13:31Z

Created page with "{| class="wikitable" |+ !Materials ! |- |Separating Gel (12,5%): |3.1 mL Polyacrylamide (40%); 4.3 mL dH2O; 2.5 mL Tris (1.5 M; pH 8.8) ''Degas!'' 50 μL Sodium dodecyl sulfate (SDS; 20%); 30 μL Ammonium Persulfate (APS; 10%); 15 μL TEMED |- |Stacking Gel (5%): |1.25 mL Polyacrylamide (40%); 6.15 mL dH2O; 2.5 mL Tris (0,5 M; pH 6,8) ''Degas!'' 50 μL Sodium dodecyl sulfate (SDS; 20%); 30 μL Ammonium Persulfate (APS; 10%); 15 μL TEMED |- |Marker..."

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{| class="wikitable"
|+
!Materials
!
|-
|Separating Gel (12,5%):
|3.1 mL Polyacrylamide (40%); 4.3 mL
dH2O; 2.5 mL Tris (1.5 M; pH 8.8)
''Degas!''
50 μL Sodium dodecyl sulfate (SDS; 20%); 30 μL Ammonium Persulfate (APS;
10%); 15 μL TEMED
|-
|Stacking Gel (5%):
|1.25 mL Polyacrylamide (40%); 6.15 mL
dH2O; 2.5 mL Tris (0,5 M; pH 6,8)
''Degas!''
50 μL Sodium dodecyl sulfate (SDS; 20%); 30 μL Ammonium Persulfate (APS;
10%); 15 μL TEMED
|-
|Marker
|Low molecular weight marker (LMW)
High molecular weight marker (HMW)
|-
|Running Buffer
|25 mM Tris-HCl
192 mL Glycine
0.1% SDS
2 L dH2O
|}

=== Procedure ===
----

* For gel electrophoresis, first a separating gel (8% - 10% - 12.5% or 15% polyacrylamide) is poured and gently covered with dH2O. The remainder of the polyacrylamide solution is set aside to monitor the polymerization reaction.
* After polymerization (~60 minutes) the dH2O is removed, the stacking gel soution (5% polyacrylamide) is added and the appropriate comb is inserted (check the proper comb thickness, slot number and wear safety goggles !)
* The stacking gel is allowed to polymerize
* The samples and marker are boiled for 5 min at 96ºC and collected with a quick spin
* In the meantime, the gel cassettes are assembled in the MiniCell Chambers according to the instructions of the supervisor and the inner chamber is filled with running buffer
* Upon removal of the comb, the sample slots are rinsed with running buffer using a syringe
* The samples (10 – 40 µl, depending on the slot size) are loaded via a Hamilton syringe
* Check that the inner chamber does not leak running buffer; fill up the outer compartment to the bottom of the gel holder with running buffer
* The separation is carried out at 100 V for about 1.5-2 h
* After the run is completed, disassemble the glass plates carefully and clean everything thoroughly. Use the separating part of the gel for i) Western Blotting or ii) Comassie staining

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