BioProtocols--DNA4

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°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

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.Washington, DC 20560. phone: 301-238-3444. fax: 301-238-3059 ...
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