PCR Protocol
| 10X PCR Buffer | 1.0 |
| MgCl2 (2.0mM) | 0.8µl |
| dNTPS mix* | 0.8µl |
| Primer-F (µg/µl) | 0.1µl |
| Primer-R (µg/µl) | 0.1µl |
| Taq (5U) | 0.1µl |
| 32P dCTP (10µci/µl) | 0.1µl |
| ddH2O | 4.5µl |
| DMSO (100%) | 0.5µl |
| SUBTOTAL | 8.0µl |
| Template DNA (50-100ng) | 2.0µl |
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 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.
Electrophoresis conditions
- Fragment Size: 150-200 bp
- 6 Watts
- 10-12 hours
- room temperature
- Fragment Size: > 200 bp
- 8 Watts
- 10-12 hours
- room temperature
Exposure
Dry gel and expose either at -80°C for 2 hours or at room temperature for 16-18 hours
