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        Mutation Detection By Single-Strand Conformational Polymorphism (SSCP)

        互联网

        1822

        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

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