Bacteriophage Plaque lifts

Tuesday, November 18, 2003

Description
Plaque transfer is typically performed using Amersham gridded, nylon, Hybond-N membranes.

Procedure
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 -20C until needed.

Recipes
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)

Supplies

Tips

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Bacteriophage Plaque lifts