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Prepping phage for a binding experiment

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Harms Lab Wiki > Phage Display > > Prepping phage for a binding experiment
  1. Day 1, afternoon: streak for single colonies of BLT5403 from glycerol stock on to LB + ampicillin plate.  Place at 37 °C.  After growth store at 4 °C.  Colonies on this plate will be good for ~1 week. 
  2. Day 2, afternoon: inoculate a 3 mL culture of LB + ampicillin from a single colony on the previous evening’s plate.  Place shaking at 37 °C overnight. 
  3. Day 3, morning: inoculate 100 mL of LB+ampicillin 1:200 with the overnight culture (500 μL).  Place shaking at 37 °C. 
  4. Grow to log phase (OD600 = 1.0), following OD600 on the lab spectrophotometer.  Usually takes 2-3 hours.  At OD600 > 1.0, the cells enter stationary phase and can dramatically lower your final phage titer. 
  5. Bring culture to 6 mM MgSO4 (600 μL of 1 M stock).  Place the BLT5403 cells at 4 °C.  These will keep for ~48 hours as hosts for new phage infections.
  6. Pull 1 mL aliquots and place in culture tubes. Add 5 μL of amplified phage to the tube.  (For libraries, you’ll want to think carefully about the ratio of phage to bacteria.  For clonal phage populations, this does not matter as much much).  Place shaking at 37 °C.  USE THE DESIGNATED LAB T7 PHAGE INCUBATOR; DO NOT USE THE COMMON GROWTH ROOMS!
  7. Wait until the cultures become clear, indicating massive bacterial death.  This usually takes ~1 hr.  Do not continue to shake at 37 °C after clarification, as this can lower final phage titer. 
  8. Add 150 μL of 4 M NaCl stock to the tube (bringing the total NaCl to ~500 mM), vortex, and then transfer to a 1.7 mL microcentrifuge tube.  Centrifuge for 10 minutes at 13,000 rpm.
  9. Recover the supernatant into a fresh 1.7 mL microcentrifuge tube.  If you care about divalent cation concentration – you probably do! – add ~0.05 g of chelex resin.  Let sit for ~15 minutes, vortexing several times. 
  10. Pull solution off the top of the chelex and place into a fresh microcentriuge.  The phage will remain infective for many months at 4 °C, however, the fused surface proteins will degrade in a protein-specific manner.  For best binding results, use the prepped phage right away.  
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