Mutation Detection By Single-Strand Conformational Polymorphism (SSCP)
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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
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.
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
