PAGE Polyacrylamide gel electrophoresis

What does gel electrophoresis do? Review n employs electromotive force to move molecules through a porous gel n separates molecules from each other on the basis of u size u charge u shape

What can polyacrylamide do that agarose can’t? Polyacrylamide has a smaller pore size than agarose, so it can n Resolve short ss DNA strands of the same length that differ in sequence. n Resolve short fragments of ds DNA that differ in length by only a few oligonucleotides. n Resolve fragments of denatured ss DNA that differ in length by only a single nucleotide. n Resolve proteins. Different gel and buffer conditions are used for these different purposes.

Acrylamide is a neurotoxin! n Acrylamide monomer in solution is toxic. u Wear gloves u Don’t inhale n Polyacrylamide gel is non-toxic.

How is polyacrylamide electrophoresis done?

Basics of Gel Formation n Acrylamide monomer polymerizes. n Bifunctional bisacrylamide makes crosslinks. n Crosslinks  matrix with pores. u = molecular sieve n Pore size decreases with increase in u total [acrylamide]. u ratio of bis to acrylamide.

What are the molecular components of a standard polyacrylamide gel? Specialty formulas: variations in crosslinkers and incorporation of different monomers; e.g., MDE - mutation detection enhancement gel.

What causes the polymerization? n Free radical reaction n Free radicals are u formed from ammonium persulfate and u accelerated by TEMED, which F catalyzes formation of free radicals n Inhibited by oxygen,so we u deaerate the polyacrylamide solution and u use a glass sandwich configuration to pour the gel.

Types of PAGE n Continuous vs. discontinous n Stacking vs. non-stacking n Denaturing vs. non-denaturing n Gradient vs. non-gradient u Buffer or acrylamide percentage n Wedge vs. uniform thickness

Our SSCP gel is n Continuous u same buffer ions in sample, gel, buffer reservoirs u constant pH n Non-stacking u same gel percentage throughout the gel n Non-denaturing u neutral pH u no denaturant molecules included n Uniform thickness n Non-gradient u same buffer concentration throughout u same gel concentration throughout