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 2 ml of overnight bacterial culture to 8 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. Alternately, approximately 106 phage from a liquid stock can be used.
  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.

Determining Phage Titer

  1. Microwave flask of Top Agar (LB + 0.8% agar) to melt. Allow to cool until handleable, then prepare 4 ml aliquots in small test tubes for as many samples as you will be handling. Place tubes in a heat block set at 55-60C.
  2. Prepare dilutions of the phage stock in LB. Typically a dilution series from 100 to 1010 in 102 increments will give one plate with a countable number of plaques. This can be easily prepared by making 1.7 ml Eppendorf tubes with 990 l LB and transferring 10 l from the higher stock, mixing, and transferring to the next sample.
  3. Combine 100 l of exponentially growing host cells and 100 l of phage dilution. Incubate unshaken at 37C for 30 minutes.
  4. Add each sample to the top agar aliquot. Mix by pipetting up and down to avoid bubbles. Pour entire contents of test tube on top of an LB plate.

Sources

Protocol adapted from Brendan Bohannan and Neerja Hajela


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Topic revision: r7 - 21 Aug 2013 - 22:53:11 - Main.AlvaroRodriguez