---+ 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) 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 50μL 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
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Topic revision: r1 - 2017-12-14 - ColinBrown