SSCP Detect DNA Mutation

DNA Mutation Detection by SSCP
Single-Strand Conformational Polymorphism (SSCP)

PCR Protocol
10X PCR Buffer 1.0ul
dNTPs mix 1.0ul
10 mM Primer-F (ug/ul) 1.0ul
10 mM Primer-R (ug/ul) 1.0ul
Taq (2U) 1.0ul
32P dCTP (10uci/ul) 0.1ul
ddH2O 2.9ul
SUBTOTAL 8.0ul
Template DNA (50-100ng) 2.0ul
# dNTPS mix (final concentration): dATP 2.5mM; dTTP 2.5mM; dGTP 2.5mM; dCTP 1.25mM

Run the PCR with standard procedure, however, it should be optimized accordingly.
SSCP Gels
Prepare 0.5x MDE gel as follows:
MDE gel 16
ddH2O 44.2ml
10X TBE 3.84ml
10% APS 256ul
TEMED 25.6ul
Pour sequencing gel format with appropriate 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 1X TEB buffer.
Heat denature samples at 94¡ãC for 5 minutes and place them on ice for 3-5 minutes. Load 2.0-4.0¦Ìl 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
Autoradiography
Dry gel and expose either at room temperature for 2 hours or at 180 centigrade for 16-18 hours.