To isolate individual phages from the total plated population
Now that you have isolated phages from your environmental sample, you are ready to start the purification process. It is possible that your environmental sample yielded more than one kind of phage. The goal of phage purification is to ensure that you end up with a phage sample that contains only one kind of phage. In other words, once purified, all the phage particles in your sample should be genetic clones of each other. This is called a “homogenous population” or a “clonal population” of phage. Having a pure phage population is very important because it will enable you to characterize your phage and discover its biological properties, including morphology, plaque size, and genome sequence. Imagine how difficult it would be to describe these properties for your phage if your sample contained many kinds of phages, each with its own morphologies and traits! Additionally, each plaque represents an individual phage that infected a single cell, and then continued to infect neighboring cells. Thus, each plaque contains hundreds to tens of thousands of viral particles! In order to purify individual phages from these picked plaques, we will need to serially dilute the samples and then perform a plaque assay to ensure a clonal population before we sequence the phages. Since we want to both have a pure stock, as well as estimate the number of phage particles per plaque, we will purify each plaque twice before amplifying the phage into a high titer lysate.
- You need to have successfully obtained phage plaques on your host, either from direct plating or enrichment
- Take notes in your lab notebook about the number of plaques on each plate. Record details about the way the plaques look. You can take photos of the plates using the scanner. Unless they have very different morphologies, choose only one plaque per plate to pick: you want to avoid isolating the same phage twice.
- SM buffer
- One labeled microcentrifuge tube per sample
- P200 pipette and pipette tips
- Label the plaques of interest with a marker on the bottom of the plate with identifiable letters or numbers to coordinate with what is recorded in your lab notebook.
Opt for a plaque that is on a plate with 2-30 plaques with isolated phages to increase your chances of selecting a single phage.
- Label the microcentrifuge tube(s) with the identifiers and today’s date. Fill each tube with 500 μL SM buffer.
- Using a sterile filter tip on the p200 pipette, gently stab the top agar the center of your plaque of interest while avoiding the bacteria around the plaque.
- Place the end of the tip into the properly labeled tube with the SM buffer. Gently tap the tip on the tube wall and pipette up and down to dislodge phage particles. Discard the tip into the biohazard waste.
- Firmly close the tube and mix by vortexing. Picked plaques can be stored at 4 C.
- Proceed to serial dilution of phages for plaque assays.
You need to have a phage stock of interest.
- SM buffer
- Microfuge tubes
- Arrange eight microfuge tubes in a rack labeled from 10-1 through 10-7. Fill each tube with 90 uL SM buffer.
- Add 10 uL of your picked plaque to the 90 uL of SM buffer in the tube labeled 10-1. Discard the pipette tip, cap the lid tightly, and vortex to mix well. This is a tenfold dilution, also referred to a 1:10 dilution.
- Transfer 10 uL of your 10-1 dilution into the 90 uL of SM buffer in the tube labeled 10-2. Discard the pipette tip, cap the lid tightly, and vortex to mix well. This is a hundredfold dilution, also referred to a 1:100 dilution.
- Continue to transfer 10 uL in successive dilution until you get to your last tube. Be sure to make use of a new, sterile filter pipette tip for each transfer.
- Continue on to the plaque assay for purification.
- 🖊️ Use a marker to divide the an LB agar plate into 9 sections, and label each dot with dilutions 0, -1 … -7 and no-phage control. Add a small label with today’s date, your name, your host bacteria and phage ID.
- 🧪 Set out as many culture tubes as plates you labeled.
- ➕ Add the appropriate bacterial host culture(s) to each of the sample tubes.
- Team Coryne:
- 250 µL of C. glu
- Team plasmid-dependent:
- 100 µL of E.coli - pRP4
- 100 µL of P. putida - pRP4
- ➕ Using a sterile serological pipette, gently add 4 mL molten top agar supplemented with CaCl2 to a single tube, and immediately suck up the top agar/sample/bacteria mixture and transfer it to the appropriately labeled plates.
- ↔️↔️ Quickly tilt the plate in multiple directions until the top agar mixture evenly coats the agar plate. Once top agar has totally coated the surface, cover the plates with the lid and leave to set.
- ⏳ Let the plates sit undisturbed for 5 minutes on the benchtop to allow the top agar to fully solidify.
- ➕ Once the top agar is set, remove the lid of the plate and carefully pipette a 10 µL drop of each phage diltution on top of the corresponding labeled plate section.
- Repeat this for every phage you’re purifying.
- ⏳ Close the lid and let the plates unidisturbed until the drops dry. It may be necessary to dry the drops underneath a lit bunsen burner.
- 🌡️ Incubate the plates overnight in the 30℃ incubator.
- Retrieve your plates from the incubator.
- Check if your phage dilutions produced single plaques and that the number of plaques decrease by approximately factors of 10 per dilution.
- Calculate the approximate titer of the plaque that you picked yesterday and record in your lab book.
Pick another plaque for each phage and repeat the spot assay for purification protocol
- Retrieve your plates from the incubator.
- Check if your phage dilutions produced single plaques and the number of plaques decrease by approximately factors of 10 per dilution.
- Calculate the approximate titer of the single plaque that you picked and record in your lab book.
- Pick a final well-isolated plaque into 500 µL of SM buffer and label this tube plaque-pure with your phage ID. ▶ Continue to Protocol 7. Preparing high titer lysates for DNA extraction