Measuring Burst Size

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  1. Prepare 10mL of media and set up cells to incubate for 1 hour in the 37C shaking incubator
    • Set up for 10^8 cells which is 12.5 ul for Black WT aliquots found in the -80 freezer
  2. After 1 hour, add phage for a total amount of 10^6 phage
    • For P21 of the pre-adapted phage, the amount needed is 8.62 ul
  3. Start the timer as soon as the phage has been added and incubate at 37C
    • The best way to do this now is to place in the shaking incubator after adding phage and when collecting samples needed do not remove flask from incubator and just take samples directly from flask
  4. Set up ten 1.5 mL microcentifuge tubes in order to collect samples
    • Prepare by adding 100 ul of chloroform into 5 of the microcentrifuge tube
    • Time periods being collected are 11 min, 13 min, 27 min, 29 min and 31 min, so label 2 tubes for each of these time periods: one will contain the chloroform treatment and the other will work as a collecting tube
  5. As soon as timer shows first time point (11 min), open shaking incubator while holding microcentrifuge tube and pipette 1 mL into the tube. Immediately vortex tube for 15 seconds, making sure a tornado is formed in the tube.
    • The chloroform treatment will kill the cells since we just want to collect the phage
  6. From this tube take 100 ul for the second empty tube with the same time label that will be used to visualize the phage
  7. Repeat steps 5-6 for remaining time periods: 13, 27, 29 and 31 minutes
  8. Conducting a phage enumeration is the best way to visualize the results, but because I am still testing out this protocol, it will be best to just use one plate with top agar and add 5 ul (or 10 ul) dots of phage in order to see if the proceedure works
    • Use the mini-dot assay and use both the undiluted and a 10^-2 dilution made by using 10 ul of the undiluted and 990 ul of PBS