P22 Phage Lysates

Protocol
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Day 1

1. Prepare fresh overnight culture of LT2 in LB.

Day 2

2. At least one hour before plating, pre-warm 8 lambda plates in 42° incubator.


3. Warm heat block with sand bath to 55°


4. Melt lambda top agar in the microwave.


5. Put bottle of melted top agar into sand bath. Allow at least a half hour for the melted agar to cool to ∼55°.


6. Dilute existing phage stock to 10³ pfu/mL in LB. If concentration is unknown or stockis old, do a series of dilutions: 10⁵, 10⁶, 10⁷, 10⁸ are good.


7. For each phage dilution, mix 0.1 mL o/n with 0.1 mL phage.


8. Mix tubes gently and incubate for 30' at 37° to ensure adsorption.


9. One tube at a time:
   • − Add ∼2.5 mL of 55° lambda top agar to tube & vortex.
   • − Pour onto plates and spread evenly by tilting and swirling gently but quickly.
   • • Tip: transfer label from tube to the plate.
   
   
10. Incubate plates ›8 hr at 37° until plaques appear. Choose a dilution with nice, isolated plaques, and store the plate at 4° overnight to use on day 3
• P22 HT plaques have turbid center.
• P22 H5 plaques are clear.


11. Start new LT2 overnight culture in LB.

Day 3

12. Prepare 125 mL baffled flask with 25 mL of "phage broth":
• LB + 0.2% glucose + 1X E salts


13. Inoculate flask with 0.1 mL of fresh overnight culture of LT2


14. Pick a "plug" with Pasteur pipette of phage plaque from the day 2 plaque plates and expel into flask.


15. Shake inoculated broth at 37° until lysis debris is evident (›24 hrs)

Day 4

16. Centrifuge phage broth at 7,000 rpm for 10 minutes to remove cellular debris. Decant into new tube and repeat if supernatant is not clear.


17. Pour supernatant (phage) into bottle and add 2 mL of CHCl₃; . Shake well, and allow to settle overnight at 4°.


18. Store in tightly sealed tubes with ∼0.5-1 mL of CHCl₃ at 4°.

##### H5 stocks can be used without titering. HT stocks should be titered. #####