Barrick Lab :: ProtocolsPhageAdsorptionRate

---+ Measuring Adsorption Rate of T7 Phage

---++ Reagents / Materials:

   * <strong>*Fresh*</strong> phage lysate (no more than <strong>*one day*</strong> old) 
      * see [[ProtocolPhageLysate][Preparing Phage Lysates]] for protocol
   * 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
   * <strong style="background-color: transparent">*For plating phages:*</strong> 
      * 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 1mL of an overnight culture of the E. coli host to 10mL of LB (+antibiotics,nsAA if needed) in a 250mL flask
   1 Allow to grow for 1h @ 37C, 200RPM until culture density is 1-2e8 cells / mL (OD ~ 0.2)
   1 Add 1e6 phages (from fresh lysate) and incubate 5min without shaking at 37C
   1 Remove 2 1mL aliquots, centrifuge one sample to pellet adsorbed phage (4000g, 1min, RT) and let the other sit at RT
   1 Plate dilutions of supernatant from pelleted sample (unadsorbed phage) and the non-centrifuged sample (total phage) 
      * plate 10uL of each dilution as described in [[stub][Determining Phage Titers]]
   1 Calculate N<sub>total</sub> and N<sub>free</sub> from plate counts, by rate equation N<sub>free</sub> = N<sub>tot</sub> e<sup>-5a</sup>; Adsorption rate a = ln(N<sub>tot</sub>/N<sub>free</sub>)/5

---++ References:

[1] Heineman, Richard H, and James J Bull. "Testing Optimality with Experimental Evolution: Lysis Time in a Bacteriophage."<em style="background-color: transparent; font-family: 'Trebuchet MS'; font-size: 12px; text-indent: -20px"> Evolution </em>61, no. 7 (2007): doi:10.1111/j.1558-5646.2007.00132.x.

-- Main.ColinBrown - 14 Jun 2017
Barrick Lab > ProtocolList > ProtocolsPhageAdsorptionRate
Contributors to this topic
ColinBrown
Topic revision: r3 - 2018-06-07 - 19:44:07 - Main.ColinBrown