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Method: Assay for Phage Containing the Beta-galactosidase Gene

April 9 1990

C. Helms


Purpose:

Special reagents:
  1. Dimethylformamide
  2. X-Gal (5-bromo-4-chloro-3-indolyl-beta-d-galactoside)
Time required:
    3 days total:

  1. Day 1: 30 minutes
  2. Day 2: 1 hour
  3. Day 3: 5 minutes
Procedure:

Day 1

  1. Pour X-Gal plates.
  2. Inoculate 5 ml LBM with indicator bacteria. Grow overnight at 37 degrees C.

Day 2

  1. Mix 0.1 ml of indicator bacteria with 3 ml of warm (50-55 degrees C) LBM top agar and pour onto X-Gal plate. Allow the top agar to set for at least 10 minutes.
  2. Dilute phage to be tested such that only 1 to 25 phage will be contained in a small drop. (e.g. 10 ul). The reason for the small number of phage is so that individual plaques can be assayed. They give a much clearer result than do confluent spots.
  3. Spot 1-10 microliters of phage dilutions onto the hardened agar. Hold for 15 minutes on the bench to let spots partially dry then incubate overnight at 37 degrees C.

Day 3

Interpretation of results:

Solutions:

References:

Sambrook J. Fritsch E.F. and T. Maniatis.(1989) Molecular Cloning A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press pp.1.83-1.84 2.43-2.53.

J. Miller. (1972) Experiments in Molecular Genetics p. 48. Cold Spring Harbor Labortory Cold Spring Harbor New York.

CRI Laboratory Manual: RFLPs Project (1989).