(r2) ProtocolsPhageLysisTiming < Lab

---+ Measuring Lysis Timing of T7 Phage

---++ Reagents / Materials:

   * Phage lysate 
      * see <span style="background-color: transparent">[[ProtocolPhageLysate][Preparing Phage Lysates]]</span><span style="background-color: transparent"> for protocol</span>
   * Overnight stock of permissive bacterial host
   * Make sure to grow in presence of 250uM nsAA if using nsAA-RS strains
   * [[stub][LB Media w/ appropriate supplements and antibiotics]]
   * 250mL flasks
   * *For plating phages:* 
      * LB Plates with w/ appropriate supplements and antibiotics
      * LB Top Agar (LB + 0.8% Agar)
      * Test tubes
      * 55C Water bath or heat block
      * 37C Incubator

---++ Procedure:

   1 Add 500uL of an overnight culture of the E. coli host to 9.5mL of LB in a 250mL flask
   1 Allow to grow for 1h @ 37C, 200RPM until culture density is 1-2e8 cells / mL (OD ~ 0.2-0.3)
   1 Add 5e7 phages and incubate 5min without shaking at 37C* (may need to be shorter for some strains?) *30C for nsAA evolved strains
   1 Dilute 100uL of culture into 900uL of media in an eppendorf tube, then transfer 10uL into 10mL fresh media pre-warmed to 37C* (10000x dilution total) (may need to adjust volumes?)
   1 Plate samples at 1min timepoints from 5-15min (may depend on strain) 
      * plate 10uL of each dilution as described in [[stub][Determining Phage Titers]]
      * Probably don't need to set up dilutions for these (?)
   1 Plot titers over timepoints; 'lysis time' is timepoint just before titer begins to increase

---++ References:

[1] Heineman, Richard H, and James J Bull. "Testing Optimality with Experimental Evolution: Lysis Time in a Bacteriophage." _Evolution_ 61, no. 7 (2007): doi:10.1111/j.1558-5646.2007.00132.x.

-- Main.ColinBrown - 14 Jun 2017
Barrick Lab > ProtocolList > ProtocolsPhageLysisTiming
Topic revision: r2 - 2017-09-13 - 13:49:32 - Main.ColinBrown