Phage Lysate Preparation

  • Definitely works with T4 and T7, should work with most E. coli phages (although lysis times and final titers may differ)

Reagents / Materials:

  • Overnight culture of a permissive host
  • BL21 usually works well for T7 and T7Δ2 (a.k.a mutator); MJH116(Amberless) with appropriate nsAA-aaRS for nsAA evolution strains
  • Frozen Stock of Phage (or fresh plating)
  • LB Media (w/ antibiotics or supplements appropriate for host)
  • 50mL flasks or test tubes
  • Shaking incubator at appropriate temp (37 or 30) *Chloroform (optional but recommended)

Procedure:

  1. Prepare overnight culture of permissive host (Amberless aaRS strains should be grown w/ 250μM nsAA in media and may need up to 24h to reach saturation)
  2. Dilute 1mL of overnight culture into 10mL fresh media (1:10 dilution), grow for 30-60min (OD ~0.2)
    • Some finicky strains (e.g. nsAA evolved strains) apparently lyse stochastically, so best to set up 8-10 lysis cultures (lyses?)
    • Can be useful to start an no-phage control (i.e. diluted host w/ no phage) for turbidity comparison w/ lysed cultures
  3. Pick a plaque from a fresh overnight plate (for clonal lysate) or scrape frozen stock with a sterile toothpick or pipet tip.
  4. Shake @~200RPM at the appropriate temperature until culture lyses completely
    • i.e. little or no turbidity compared to no-phage control; will usually see bits of aggregated cell debris also
  5. Add 50-100uL of chloroform and vortex
  6. Spin 5min @ 5000RPM
  7. Transfer lysate (supernatant) to a clean tube
    • For long-term storage @ 4C, add 50μL chloroform to prevent bacterial growth
    • For long-term storage @ -80C, add glycerol to 20% and mix well
  8. Determine phage titer Protocol: Measuring Phage Titers

-- Main.ColinBrown - 14 Dec 2017

 Barrick Lab  >  ProtocolList  >  ProtocolsPhageAdsorptionRate  >  ProtocolPhageLysate

Topic revision: r1 - 14 Dec 2017 - 22:00:57 - Main.ColinBrown
 
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