![]() Carefully remove the gels from the casting stand and then from their green frames.Ģ. If air bubbles become trapped below the comb, remove the comb and reposition it.ġ. (The Bio Rad logo on the comb should be facing you.) Adding the comb will force some solution out of the gel, but this is fine. Carefully, but quickly, lower the comb into position, being careful not to introduce air bubbles.Add enough stacking solution until it just reaches the top of the small plate. Use a transfer pipette to pipette the stacking gel on top of the resolving gel between the two glass plates.Mix the contents by gently inverting the tube twice. Add 30 μL 10% APS and 7.5 μL TEMED to the stacking gel acrylamide mixture.Mix the acrylamide solution, pH 6.8 Tris buffer and water, as shown in the chart above. SAFETY NOTE: Be sure you are wearing goggles when pouring the stacking gels. When polymerization is complete, remove the water from the top of the resolving gel by tilting the gel to the side and using a paper towel or Kimwipe to wick out the water.(You can also check the remaining polyacrylamide in the transfer pipette to see if it has polymerized.) A sharp new interface then forms between the two layers, indicating that polymerization is complete. You will note that the interface between the polyacrylamide and water overlay disappears temporarily while the gel polymer- izes. Allow the gel to polymerize, which takes ~15-20 minutes.The water layer ensures that the polyacrylamide gel will have a level surface once it polymerizes. Using a transfer pipet, add deionized water so that it gently flows across the surface of the polyacrylamide mixture.(You will know that the acrylamide has polymerized when you can no longer push the acrylamide out of the pipet.) Draw up any remaining acrylamide into the transfer pipet. Working quickly, use a plastic transfer pipette to fill the space between the two plates until the resolving gel solution reaches a height just above the green clamps on the gel casting frame.Cover the tube immediately after you aliquot this reagent. Once again, gently mix in the catalyst trying not to introduce air bubbles. Oxygen inhibits acrylamide polymerization. Gen- tly mix the solution, trying not to introduce air. To the resolving gel mixture, add 100 μL of a 10% ammonium persulfate (APS) solution.Oxygen inhibits polymerization of acrylamide gels. Mix the ingredients gently, trying not to introduce air. Mix the acrylamide solution, pH 8.8 Tris buffer and water, as shown in the chart above. Prepare ONLY the resolving gels at this time.Label two 15 mL conical tubes “Resolving gel” and “Stacking gel”.If the gel leaks, disassemble the frame, dry the plates and go back to step 3.Ĭatalysts should NOT be included into the mixture until you are ready to pour the gels!! Use paper towels or tissues to absorb any residual water. Pour the water out by holding the entire cast- ing platform over a liquid waste container or sink. If the glass plates hold water and don’t leak, you are ready to make the gels. Check to see if the assembled plates in the casting stand are sealed properly by pipetting a small amount of deionized water into the gap between the plates. ![]() Repeat steps 1-5 to prepare a second gel in the casting frame.
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