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Mutation Detection

By Single-Strand Conformational Polymorphism (SSCP)

PCR Protocol

10X PCR Buffer 1.0
MgCl2 (2.0mM) 0.8l
dNTPS mix* 0.8l
Primer-F (µg/µl) 0.1l
Primer-R (µg/µl) 0.1l
Taq (5U) 0.1l
32P dCTP (10µci/µl) 0.1l
ddH2O 4.5l
DMSO (100%) 0.5l
SUBTOTAL 8.0l
Template DNA (50-100ng) 2.0l

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

SSCP Gels

Prepare 0.5x MDE gel as follows:

MDE gel 16.0ml
ddH2O 44.2ml
10X TBE 3.84ml
10% APS 256µl
TEMED 25.6µl

Pour sequencing gel format with appropriate sharkstooth comb. Gel will polymerize in about 1 hour.

Loading Buffer

  • 95% formamide
  • 10mM NaOH
  • 0.025% Bromophenol Blue
  • 0.025% Xylene Cyanol

Run gel in 0.6X TEB buffer.

Heat denature samples at 94C for 5 minutes and place them on ice for 3-5 minutes. Load 2.0-4.0l per sample. Include non-denatured controls.

Electrophoresis conditions

  1. Fragment Size: 150-200 bp
    • 6 Watts
    • 10-12 hours
    • room temperature
  2. Fragment Size: > 200 bp
    • 8 Watts
    • 10-12 hours
    • room temperature

Exposure

Dry gel and expose either at -80C for 2 hours or at room temperature for 16-18 hours.


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This document was created for the neurogenetics laboratory run by P. H. Reddy, Ph.D. in the NSI of OHSU. Page last updated: 19 Sept 2001.