(r1) Barrick Lab :: ProtocolPhageLysate

---+ 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 T7&Delta;2 (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/ 250&mu;M nsAA in media and may need up to 24h to reach saturation)
   1 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
   1 Pick a plaque from a fresh overnight plate (for clonal lysate) or scrape frozen stock with a sterile toothpick or pipet tip.
   1 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
   1 Add 50-100uL of chloroform and vortex
   1 Spin 5min @ 5000RPM
   1 Transfer lysate (supernatant) to a clean tube 
      * For long-term storage @ 4°C, add 50&mu;L chloroform to prevent bacterial growth
      * For long-term storage @ -80°C, add glycerol to 20% and mix well
   1 Determine phage titer [[ProtocolsPhageTiters][Protocol: Measuring Phage Titers]]

-- Main.ColinBrown - 14 Dec 2017

Barrick Lab > ProtocolList > ProtocolsPhageAdsorptionRate > ProtocolPhageLysate

Contributors to this topic

JeffreyBarrick, ColinBrown

Topic revision: r1 - 2017-12-14 - 22:00:57 - Main.ColinBrown