Friday, June 04, 2004
Description RAPD ( Randomly Amplified Polymorphic DNA) analysis with 'P. infestans' Procedure Reaction conditions: 25 ul per reaction sterile di water 16 ul 1O X buffer 2.5 ul 100 mM KCI 200 mM Tris-HCI (pH 8.8) 100 mM (NH4)2SO4 20 mM MgSO4 1% Triton X-100 100 X bovine serum albumin 0.25 ul (10 mg/ml, nonacylated) 1.25 mM dNTP 2.0 ul 1.25 mM each of dATP, dCTP, dGTP, and dTTP 5 units/ul Taq DNA polymerase 0.2 ul **2 ng/ul DNA stock (in 10 mM Tris 7.5, 0.1 mM EDTA (either bulk DNA or individual progeny)) 2.0 ul **10 uM primer (in 10 mM Tris 7.5, 0.1 mM EDTA) 1.0 ul **usually one or more of these components are first added to the well, and then 25 ul of a "master mix" is added. Cycling conditions (for 96-well MJ research machine using microliter plates; double times for Perkin Elmer 480 machine using standard 0.5 ml tubes) 94 C, 15 sec. 1X 94 C, 30 sec; 35 C, 30 sec; 72 C, 60 sec. 36 X 72 C, 2.5 min. 1X cool to 10 C, then shut off Electrophoresis Run on 1.6% gelin TBE at -5 V/cm (4.8 g per 300 ml) Load 6-12 ul depending on well size (too much will smear) Run until bromophenol blue tracking dye is 1 cm from end of gel Stain in 0.5 ug/ml ethidium bromide (in water), 30-60 minutes. Destain in water 10 to 30 minutes, photograph Recipes Bulked Segregant Analysis Michelmore et al. PNAS 88:9828-9832 (1991) Supplies Tips (责任编辑:泉水) |