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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.
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