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
Run gel in 1X 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
Autoradiography
Dry
gel and expose either at room temperature for 2 hours or at 180 centigrade for 16-18 hours.
Other protocols for mutation/SSCP
Protocol:
Mutation Detection by SSCP PCR.
A protocol for mutation detection by single-strand conformational polymorphism (SSCP) by PCR from the
neurogenetics laboratory in the Neurological Sciences ...
www.ohsu.edu/nsi/faculty/reddyh/lab/protsscp.html
Step by Step SSCP Travis
Glenn Laboratory of Molecular Systematics ...
Step by Step SSCP. Travis Glenn. Laboratory of Molecular Systematics. Smithsonian Institution.
www.uga.edu/srel/DNA_Lab/SSCP'96V2.rtf
Protocol: Mutation Detection
by SSCP PCR.
A protocol for mutation detection by single-strand conformational polymorphism (SSCP) by PCR from the neurogenetics laboratory
in the Neurological Sciences ...
www.ohsu.edu/nsi/faculty/reddyh/lab/protsscp.html