You will need:
Denaturing buffer (1.5M NaCl, 0.5M NaOH)
Neutralising buffer (1.5M NaCl, 0.5M Tris.Cl, pH 7.2, 1mM EDTA)
20 x SSC (3M NaCl, 0.3M sodium citrate, pH7.0)
1) Place Hybond-N, 82mm membrane circles onto the surface of agar plates carrying bacteriophage infected bacteria, gridded face upper most, and mark the orientation with a syringe needle in an assymetric manner.
2) After approximately 1 minute, gently remove the membrane with forceps and place plaque side up, onto Whatman 3MM filter paper soaked in denaturing buffer.
3) After 1 minute, remove the membrane and place, again plaque side up, onto Whatman filter paper soaked in neutralizing buffer for 5 minutes.
4) After neutralisation, remove the membrane, wash for at least 2 minutes in 2 x SSC and air dry.
5) Covalently link DNA to the membrane by illumination, at 254nM, with UV radiation by placing the membrane face-down on the surface of a transilluminator for 1 minute.
Membranes can be stored between pieces of Whatman 3MM filter paper at -20°C until needed.
Dr. Simon Dawson,
Department of Biochemistry,
University of Nottingham,
The Medical School ,
Clifton Boulevard ,
Tel: +44 (0)115 9249924 ex.44787,
FAX: +44 (0)115 9422225,
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