Phage Lysate Preparation

Optimized for the production of high titer T4 lysates (1010-1012 PFU/ml). It is also suitable for phages T5 and T6 but tends to give lower titer lysates (107-109 PFU/ml).

  1. Prepare overnight culture of permissive host.
  2. Prepare a fresh plating of phage using rich media. (M9 supp plates and top agar are optimum media, but LB plates can be used.)
  3. Transfer 1 ml of overnight bacterial culture to 4 ml of rich media in a 50 ml flask. (M9 supp is optimal, but LB will also work.)
  4. Pick a single phage plaque with a toothpick and drop into culture. Titer can sometimes be improved for phages other than T4 if you wait 30 min after diluting bacterial culture before inoculating with phage.
  5. Shake at 37C. The culture should completely lyse in 6-8 hours (it will still be slightly turbid due to cell debris).
  6. Add 10 drops of chloroform and mix thoroughly to kill remaining bacteria.
  7. Spin at 5000 rpm for 5 min to remove cell debris. If the lysate is of very high titer it should appear slightly milky.
  8. Decant supernatant (including a few drops of chloroform).
  9. Titer phage on rich media. Be careful to let chloroform settle to the bottom of the tube before removing lysate.
  10. Store lysates at 4C as is, and/or in 20% glycerol at -80C.

Sources

Protocol adapted from Brenden Bohannan and Neerja Hajela
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Topic revision: r3 - 12 Jan 2009 - 22:18:43 - Main.JeffreyBarrick
 
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