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Acrylamide Polymerization A Practical Approach

where %T refers to % (w/v) of total monomer (acrylamide + bis) in solution. A higher concentration of monomer results in faster polymerization. Therefore, changing from 5% gels to 30% gels will probably allow a reduction of 2050% in the concentration of initiators.

Polymerization Protocols
There are 2 major initiator formulations for acrylamide polymerization. The first, for chemical polymerization, is used for SDS-PAGE and DNA sequencing. Chemical polymerization employs ammonium persulfate and TEMED as initiators. The second, for photochemical polymerization, is used primarily for horizontal electrofocusing gels. Photochemical polymerization calls for riboflavin as well as ammonium persulfate and TEMED. Riboflavin phosphate can be substituted for riboflavin. Riboflavin phosphate is often preferred for its greater solubility.

Preparation for Polymerization
1. Prepare 10% ammonium persulfate shortly prior to use (prepare fresh daily). TEMED is used undiluted. Prepare 0.1% riboflavin (or riboflavin phosphate, which is more soluble) if photopolymerization will be performed.

2. Combine buffer stock solution, monomer stock solution, and water in the appropriate proportions in an Erlenmeyer flask. Since stock solutions are usually stored at 4C, the gel solution should be allowed to warm to room temperature before degassing.

3. Prepare the gel casting mold, i.e., plates, spacers, and clamps for gel casting. Be sure they are neither hot nor cold.

4. Once the gel solution is prepared and brought to room temperature (2325C), degas the solution under a vacuum of 125 torr or better for 15 min at room
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