May 25, 2026
Triple plaque purification to generate clonal phage lysates
- 1University of Michigan
- Duhaime Lab
Protocol Citation: Melissa Duhaime 2026. Triple plaque purification to generate clonal phage lysates. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2b63l1y9/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 17, 2017
Last Modified: May 25, 2026
Protocol Integer ID: 8291
Keywords: triple plaque purification, clonal phage lysates use, clonal population of phage, plaque, phage, lysate, clonal population
Abstract
Use to purify plaques to attain a lysate containing a clonal population of phages.
Purification Rounds 1-3
Start with an overnight agar overlay plate that shows plaque formation.
Using a 200 ul pipette tip, pick a plaque from the original host screening plate that plaques were discovered on. Choose a plaque that is similar in morphology and size to the majority of the other plaques on the plate.
Mark on the plate which plaque was picked for future reference.
Vortex the pipette tip in 1 mL of phage buffer. Let the sample stand for at least 30 minutes. Vortex again before plating sample.
00:30:00 bringing phage into solution
Plate dilutions of 10-2 - 10-6. This will help to ensure countable data after incubation.
Incubate overnight at room temperature.
16:00:00 overnight incubation (can be 16-28 hours)
Pick a plaque from overnight plates for Purification Round 2.
Repeat steps above for one more round of purification. This is purification 3.
16:00:00 overnight incubation (can be 16-28 hours)
Generate clonal lysate
Pick plaque from Purification 3 plate using a 200 µl pipette tip. Vortex the pipette tip in 1 mL of phage buffer. Let the sample stand for at least 30 minutes. Vortex again before plating sample.
00:30:00 bringing phage into solution
Using a dilution that will lyse 95% of the host lawn, incubate for 24 hours at RT.
24:00:00 overnight incubation
Collect Lysate
Identify plates that are 95% lysed. They can be as low as 70%, but completely lysed plates should not be used. Hold plate at an angle, so as not to disturb top agar. Gently pipette 5 mL of phage buffer onto plate. Place on platform shaker at 70 rpm for 30 min to 3 hr at room temperature.
00:30:00 can be 30 mins to 3 hrs
Phages should now be suspended in buffer. Use a serological pipette to extract liquid into a 15 mL conical tube. If there is solid matter in the sample, centrifuge the sample at 3220 g for 5 min and extract the supernatant.
Using a 5-10 mL syringe, filter the sample into a new 15 mL conical tube using a 0.8/0.2 um filter (blue and green).
Determine titer of lysate
Plate plaque assays on corresponding hosts, at dilutions 10-1 - 10-9.
Incubate plate for 24 hours.
24:00:00 incubation
Count plaques the following day. If there are fewer than 3 x 10-9 phage / mL, make multiple plate lysates of that phage and concentrate the combined lysate with PEG or Amicon filtration cartridges.