Gill:Spot complementation test

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Spot complementation test
Spot complementation test

Rev. 2015-03-05

Adapted from: Hendrickson & McCorquodale 1971, Genetic and Physiological Studies of Bacteriophage T5: I. An Expanded Genetic Map of T5. J Virol 7: 612-618, PMID 16789131.

The purpose of this assay is to determine if mutations in a set of phages are present in the same gene or in different genes; the phages are tested pairwise. Host cells that are not permissive for phage growth are co-infected with two different phage mutants. If the two phages have defects in different essential genes, then the co-infected cell will contain one functional copy of each gene, and this in trans complementation will allow the phages to lyse the cell and form a plaque. If the two phages have defects in the same gene, they will not be able to complement and no plaques will form.

Materials

  • Agar plates
  • T-top agar (in 4 ml aliquots at 48-50 ºC)
  • Fresh overnight or over-day culture of non-permissive host cells
  • Stocks of phage mutants, adjusted to ~107 PFU/ml in lambda diluent or LB

Procedure

  1. Label the bottom(s) of the plates with each pairwise combination of phage you will test. You should be able to fit 6 phage pairs on each plate.
  2. Add 100 µl of the bacterial culture to a 4 ml T-top aliquot, mix and pour over the surface of a plate. Allow the lawn to set for a few minutes.
  3. Apply a 10 µl drop of one phage from each test pair to the plate, allow the plate to air-dry for ~10 min or until the drop is adsorbed into the lawn.
  4. Apply a 10 µl drop of the second phage from each test pair to the plate to form two partially overlapping spots for each pair. Allow the plate to air-dry for ~10 min or until the second drop is adsorbed into the lawn.
  5. Incubate the plates overnight at 37 ºC and observe the plates for clearing. Clearing in the overlap region where cells were mixedly infected indicates complementation.
    Controls: spot 10 µl of the wt phage to a lawn as a positive control, and spot each phage against itself as a negative control.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

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