PAGE Polyacrylamide gel electrophoresis. What does gel electrophoresis do? Review n employs electromotive force to move molecules through a porous gel.

PAGE

Polyacrylamide gel electrophoresis

What does gel electrophoresis do?Review

employs electromotive force to move molecules through a porous gel

separates molecules from each other on the basis of size charge shape

What can polyacrylamide do that agarose can’t?

Polyacrylamide has a smaller pore size than agarose, so it can

Resolve short ss DNA strands of the same length that differ in sequence.

Resolve short fragments of ds DNA that differ in length by only a few oligonucleotides.

Resolve fragments of denatured ss DNA that differ in length by only a single nucleotide.

Resolve proteins.Different gel and buffer conditions are used for these different

purposes.

Acrylamide is a neurotoxin!

Acrylamide monomer in solution is toxic. Wear gloves Don’t inhale

Polyacrylamide gel is non-toxic.

How is polyacrylamide electrophoresis done?

Basics of Gel Formation

Acrylamide monomer polymerizes. Bifunctional bisacrylamide makes crosslinks. Crosslinks matrix with pores.

= molecular sieve Pore size decreases with increase in

total [acrylamide]. 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?

Free radical reaction Free radicals are

formed from ammonium persulfate and accelerated by TEMED, which

catalyzes formation of free radicals Inhibited by oxygen,so we

deaerate the polyacrylamide solution and use a glass sandwich configuration to pour the

gel.

Types of PAGE Continuous vs. discontinous Stacking vs. non-stacking Denaturing vs. non-denaturing Gradient vs. non-gradient

Buffer or acrylamide percentage Wedge vs. uniform thickness

Our SSCP gel is Continuous

same buffer ions in sample, gel, buffer reservoirs constant pH

Non-stacking same gel percentage throughout the gel

Non-denaturing neutral pH no denaturant molecules included

Uniform thickness Non-gradient

same buffer concentration throughout same gel concentration throughout