---+ 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
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Topic revision: r2 - 2017-09-13 - ColinBrown