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Titering a Phage Library

Adapted from Current Protocols in Molecular Biology and the Stratagene® instruction manual for HybriZAP Two-Hybrid cDNA Gigapack® Cloning Kit.


Objective:

To determine the number of plaque forming units per mL (pfu/mL), which is a measure of the concentration of infectious phage. This number can then be used to dilute the phage allowing maximum plaques without plaque superimposition.


Required Materials

  • Recombinant DNA phage library (aliquot from -70°C or at 4°C for 2 months or less).
  • Host bacteria- XL1-Blue MRF' -freshly streaked on an LB-Tet plate.
  • 86mm NZYM top agarose.
  • Suspension Media (SM)

Procedures

  1. Pick a colony of XL1-Blue MRF' from the LB-Tet plate and grow to saturation in 5 mL of NZYM broth.
  2. Melt NZYM top agarose in microwave and place in waterbath to equilibrate at 45-50°C.
  3. Make 4 serial dilutions (1:100) of the phage library in SM: 1:100; 1:10,000; 1:1,000,000; & 1:100,000,000.
  4. Label 6 tubes. One for each dilution of phage, one for no phage (optional control plate), and one for no cells (optional control plate).
  5. Label as in 4 above and pre-warm (37°C) 6 NZY agar plates.
  6. Add 0.3 mL of saturated XL1-Blue MRF' cells in NZYM broth to each labeled tube except the control for no cells to this tube add 0.3 mL fresh NZYM broth.
  7. Add 100 µL of each dilution to the appropiately labeled tube of cells. Add 100 µL of SM to the two control tubes.
  8. Incubate at room temperature while gently shaking for 20 minutes to allow the bacteriophage to adsorp to the bacteria.
  9. Incubate at 37°C for 10 minutes. During this time the adsorped bacteriophages inject their DNA into the host bacteria.
  10. Add 3.0 mL of NZYM top agarose to each tube, briefly mix by vortexing, and pour onto 100mm NZY agar plates. Spread the agarose across the plate by tilting the plate gently.
  11. After all the top agarose/cells have been poured and set, move the plates to the 37°C incubator for 8-12 hours. Don't allow the plates to incubate too long. The plaques will grow quickly and superimpose.
  12. Count the number of plaques per plate for one of the dilutions to determine the pfu/µl of the stock library.

The plates can be stored at 4°C for a week or more.


Recipes

  • 5.8 g NaCl
  • 2 g MgSO4*7H2O
  • 50 mL 1 M Tris-HCl (pH 7.5)
  • 0.1 g gelatin (Difco)