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 @ 4°C, add 50μL chloroform to prevent bacterial growth
   • For long-term storage @ -80°C, add glycerol to 20% and mix well
8. Determine phage titer Protocol: Measuring Phage Titers

-- Main.ColinBrown - 14 Dec 2017