PCR Protocol

10X PCR Buffer	1.0
MgCl2 (2.0mM)	0.8µl
dNTPS miundefined	0.8µl
Primer-F (µg/µl)	0.1µl
Primer-R (µg/µl)	0.1µl
Taq (5U)	0.1µl
32 P dCTP (10µci/µl)	0.1µl
ddH2 O	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.

SSCP Gels

Prepare 0.5x MDE gel as follows:

MDE gel	16.0ml
ddH2 O	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

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 -80°C for 2 hours or at room temperature for 16-18 hours