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How to Pour Gradient Gels

A gradient gel is a mixture of two concentrations of acrylamide gel which are poured in such a way as to create a smooth transition from a high concentration of acrylamide at the bottom to a lighter concentration at the top. They are generally pumped into cassettes which sandwich the gel between two panes of glass. Gradient gel cassettes are used in the laboratory to determine protein weights and the migration properties of heavy organic molecules.

Things You'll Need

Gel cassette
2-chamber gradient maker
Stirring stand
2 stirring bars
Gradient gel solution
Ammonium persulfate (APS)
Tetramethylethylene diamine (TEMED)
Water saturated n-Butanol

Instructions

Setting up the Apparatus
- 1
  Assemble the gel cassette according to the manufacturer's instructions, ensuring that the bottom of the cassette is completely sealed.
- 2
  Place the gradient maker on a stirring stand at a level higher than the top of the cassette. Place a stir bar in each chamber. Close all outlets and place the outlet tube in the top of the cassette so the gel will flow into the top center of the cassette.
- 3
  Fill the nonoutlet cylinder of the gradient maker with a gel solution mixed to the lowest desired concentration. Open the stopcock between the two cylinders to clear out any air bubbles and close the stopcock. Fill the outlet cylinder with gel solution mixed to the highest desired concentration.
Pouring the Gel
- 4
  Mix 1 mL APS and 0.1 mL TMED per 100 mL of gradient gel into each cylinder.
- 5
  Open the stopcock between the cylinders. Open the outlet to a flow rate which will empty the cylinders in 5-8 minutes. Stir the contents of the outlet cylinder constantly until both cylinders are completely drained.
- 6
  Overlay the gel with water saturated n-Butanol.

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