Phage-pharming (how to make the phage stocks)
Thanks to Brendan Bohannan for the following protocol on preparing phage lysates!
© 1999 by Brendan Bohannan
Protocol for Phage Lysate Preparation
Note: this protocol is optimized for the production of high-titer T4 lysates (10e10 -10e12 pfu per ml). It can also be used for phages T2, T7, T5 and T6 but titers tend to be lower with these phages (10e7 - 10e9 per ml).
- Prepare overnight culture of permissive host (e.g., REL607 for T4,T5,T2, T7; LS371 for T6).
- Prepare a fresh plating of phage using rich media (M9supp plates and top agar are optimum media, but phage plates and soft agar can be used, or even LB). Phage should be diluted such that plaques are not touching.
- Transfer 1 ml of overnight culture to 4 ml of rich media in a 50-ml flask (M9supp is optimum media, but LB will also work).
- Stick a toothpick into an isolated plaque (use sterilized forceps to handle toothpick). Drop the toothpick into the host culture. Titer can sometimes be improved for phages other than T4 if you wait 30 minutes after diluting bacterial culture before adding toothpick.
- Shake at 37C. The culture should completely lyse in 6-8 hours (it will still be slightly turbid due to cell debris).
- Add 10 drops of chloroform and mix thoroughly to kill remaining bacteria.
- Spin at 5000 rpm for 5 minutes to remove cell debris. If the lysate is of very high titer it should appear slightly "milky" (similar to a DM25 bacterial culture).
- Decant supernatant (including a few drops of chloroform).
- Titer phage on rich media (M9 is ideal, others can be used, see above). Be careful to let chloroform settle to bottom of tube before removing aliquot of lysate for titering.
- Store lysates at 4C as is, and/or in 20% glycerol at -80C.
Recipe for M9supp Media (makes 100 ml)
|10X M9 Salts||10 ml|
|10% Glucose||5 ml|
|0.1M MgSO4||1 ml|
|20% Casamino Acids||5 ml|
|10-mg-per-ml B1||0.02 ml|
|10-mg-per-ml Tryptophan||0.02 ml|
|sterile dH2O||79 ml|
M9 Agar is same as above but with 15g agar per liter; M9 Top Agar is same as above but with 7 g agar per liter. Autoclave agar and dH2O, then add sterile supplements.
Recipe for 10X M9 Salts (makes 1 L)
Autoclave; add 1 ml of 1M CaCl2.