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From 2012.igem.org

SDS-polyacrylamide and native gel electrophoresis

Reagents & Materials

Procedure

• Wear gloves
• Clean glass slides with MQ and dry

running gel

1. Prepare running gel mix in a small beaker for 2 gels:
• 4 ml MQ
• 3.3 ml 30% acrylamide mix
• 2.5 ml 1.5 M TRIS pH 8.8
• 0.1 ml 10% SDS
• 0.1 ml 10% ammonium persulphate
2. Places glass slides in holders and tighten in rack.

polymerization:

3. Add 4 ul TEMED to the running gel mix, mix and pipet the gel into the slides until the green edge.
4. Add a layer of iso-propanol on top of the polymerizing gel to equalize.

Stacking gel

1. When the running gel has fully polymerized, remove the remaing iso-propanol with a tissue
2. Prepare stacking gel mix in a small beaker for 2 gels:
• 1.4 ml MQ
• 0.33 ml 30% acrylamide mix
• 0.25 ml 1 M TRIS pH 6.8
• 0.02 ml 10% SDS
• 0.02 ml 10% ammonium persulphate
3. Add 2 ul TEMED to the stacking gel mix, mix and pipet the gel onto the running gel.
4. Place comb
5. After the stacking gel has fully polymerized, the gels can be stored at 4 degree Celcius in a box with wet tissues.

Sample preparation

Protein denaturation for SDS-PAGE:

1. Dependent on the protein concentration, typically 15 ul of sample are transferred to a 1.5 ml tube
2. Pipet 5 ul of SDS-PAGE loading buffer 5X in the caps of the tubes
3. Spin tubes briefly
4. Heat samples and a protein marker in a thermobloch 10' at 99 degree Celcius
5. Spin down samples briefly