By Single-Strand Conformational Polymorphism (SSCP)
Note: Synthesize primers 21-30 nt in length for products of 200-350 bp.
Use standard PCR conditions, however the Tm has to be calculated from the specific primer pair.
*dNTPS mix (final concentration): dATP 2.5mM; dTTP 2.5mM; dGTP 2.5mM; dCTP 1.25mM
Prepare 0.5x MDE gel as follows:
Pour sequencing gel format with appropriate sharkstooth comb. Gel will polymerize in about 1 hour.
Run gel in 0.6X TEB buffer.
Heat denature samples at 94°C for 5 minutes and place them on ice for 3-5 minutes. Load 2.0-4.0µl per sample. Include non-denatured controls.
Dry gel and expose either at -80°C for 2 hours or at room temperature for 16-18 hours.
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