P22 Phage Lysates
(Modified by Doug Huseby from Renée Dawson)
- Prepare fresh overnight culture of LT2 in LB.
- At least one hour before plating, pre-warm 8 lambda plates in 42° incubator.
- Warm heat block with sand bath to 55°
- Melt lambda top agar in the microwave.
- Put bottle of melted top agar into sand bath. Allow at least a half hour for the melted agar to cool to ∼55°.
- 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.
- For each phage dilution, mix 0.1 mL o/n with 0.1 mL phage.
- Mix tubes gently and incubate for 30' at 37° to ensure adsorption.
- 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.
- 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
Start new LT2 overnight culture in LB.
- P22 HT plaques have turbid center.
- P22 H5 plaques are clear.
- Prepare 125 mL baffled flask with 25 mL of “phage broth”:
- LB + 0.2% glucose + 1X E salts
Inoculate flask with 0.1 mL of fresh overnight culture of LT2
Pick a “plug” with Pasteur pipette of phage plaque from the day 2 plaque plates and expel into flask.
Shake inoculated broth at 37° until lysis debris is evident (›24 hrs)
- 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.
- Pour supernatant (phage) into bottle and add 2 mL of CHCl3; . Shake well, and allow to settle overnight at 4°.
- 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.
Last Update: Thursday, 19-Jun-2014 11:54:37 PDT
This page has been viewed 197 times.
eckofoid at ucdavis.edu