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)

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