Method: Picking Single Phage Plaques

June 7 1990

Srini Ramachandra

Purpose:

To isolate single bacteriophage lambda plaques to be used for making lysates.

Time required:

30 minutes

Procedure:

1. Place 0.1 ml of lambda diluent (10 mM Tris-HCl pH8.0 2 mM MgCl2) into a sterile 15 ml snap-cap tube.
2. Using a sterile short pasteur pipet attached to a Pipet-aid or to a rubber bulb stab through an individual well isolated plaque. Apply mild suction so that the plaque together with the underlying agar is drawn into the pipet.
3. Gently expel the agar from the pipet into 0.1 ml of lambda diluent in a sterile tube. Let stand at room temperature for at least 20 minutes to allow the phage to diffuse out of the agar.
4. These phage plugs can be stored at 4deg.C for several weeks without loss of viability as long as they do not dry out or can be used immediately to make plate lysates. Follow the plate stock procedure adding 0.1 ml of an LE392 overnight culture directly into the tube containing the plaque.

   Note: Because the lambda phage can diffuse considerable distances through the top agar layer pick freshly plated well separated plaques.

   References: none

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