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From 2010.igem.org

Wear gloves at all times!

Requirements:

• 30% acrylamide(Biorad) NEUROTOXIN!
• 4X separation gel buffer: 1.5M Tris.HCl, 0.4% SDS pH6.8
• 4X stacking gel buffer: 0.5M Tris.HCl, 1.92M glycine, 1% SDS water saturated isobutanol
• TEMED( in yellow cabinet )
• 10% APS (Ammonium persulfate) ; prepare 10 ml and keep cold in fridge

Hoeffer Mighty gel system Casting of the gel:

• Per gel take one small glass plate with 'ears', one big white ceramic plate, two grey spacers with perpendicular plastic bits (0.75 mm), and one white comb (0.75 mm)
• Clean the plates etc with soap, rinse with demiwater and ethanol, and dry
• Assemble the system in the gel casting holder. Mark the line of separation/stacking
• Mix the separation gel in 10 ml plastic tube:

                              for 2 gels
Seperation gel (0.75 mm)               12.5%            16%    
   30% acrylamide                      4 ml             5.1 ml
   4X separation buffer                2.4 ml           2.4 ml
   MQ                                  3.2 ml           2.1 ml
   10% APS                             28 μl            28 μl
   TEMED                               28 μl            28 μl

• Pipet the separation gel mix immediately in between the glass plates until the marked line is reached
• Pipet water-saturated isobutanol on top of the polymerizing separation gel
• Let the separation gel polymerize completely before preparing the stacking gel
• When the gel is polymerized, discard the isobutanol and wash the gel with water
• Mix the stacking gel in a 10 ml tube:

                               for 2 gels
   mQ                               1.92 ml
   4X stacking buffer               0.83 ml
   30% acrylamide                   560 μl
   10% APS                          14 μl
   TEMED                            7 μl

• Pipet the stacking gel mix immediately on top of the separation gel and place the comb without air bubbles. Mark the teeth of the comb with a marker
• Let the gel polymerize (5-30 min); check also the remaining gelmix in the tube
• Disassemble the gel casting holder and take out the gel/plates
• Use the gel immediately or seal the gel in plastic seal bags and store at 4 °C

Assembly of the Tris-Glycine gel in the electrophoresis unit

• Take the electrophoresis unit of the Hoeffer system and place it next to a power unit
• Dilute 10X and pour 1X electrophoresis buffer in the container( roughly 1 cm high)
• Place gel in the buffer without air bubbles under the gel
• Use the red clamps to place the gel tightly in the unit
• Pour 1X electrophoresis buffer in the chamber so that the top of the gel is immersed
• Take the comb out of the gel and rinse the wells using a hooked needle and syringe

Runninge of the gel

• Prepare the samples in 1X SDS sample buffer (NOT nucleic acid loading buffer)
• Boil the samples for 5 min and spin down
• Pipet the samples and protein marker carefully into the wells
• Place the electrode cap on the unit and press lightly.Put the other side of the electrode cables in the correct holes of the power unit
• Switch on the power unit and run until the blue front is appr. 1 cm from the end of the gel. Alternatively, use the prestained protein marker to identify the exact point of stopping
• Disassemble the electrophoresis unit and take the gel
• Take one plate off and cut one corner away for positioning purpose
• Carefully bring the gel into a clean staining container using some demi water and/or spacers
• Stain the gel with CBB or silver