I've been attempting to isolate bacteriophages from water samples, but unlike my usual observations, this particular phage is forming very minute plaques. Could this indicate something? Additionally, apart from sequencing, is there a method to determine whether the phage is lytic or lysogenic?
Some phage do form small plaques so that may be fine. But perhaps altering the media conditions or the temperature can give you larger plaques.
The usual sign of a lysogenic phage is that the plaque is turbid, many cells growing inside the plaque. You might just inspect the plaques under a low power microscope since you have small plaques (or zoom in close with a good camera) and see if they are truly clear or have some turbidity.
Atif Khan sir, If that’s the case then how can I estimate the size of the genome. Will running it on 0.8% agarose gel give any useful insights.
Estimating size of phage genome on agarose gel is not possible since majority of phage genome size varies from 50- 150 KB for which DNA ladder is not available. Teh other possible way is to run PFGE.
Second part of your question, if you want to know your phage is lytic or lysogenic, pick your phage and amplify ti to high titer and do a spot test on the lawn of host. If the zone of lysis is clear it is lytic, otherwise lysigenic.
Actually if you have sufficient DNA you can do a few restriction digests of the phage genome and add up the fragment sizes. If you do this with a few different enzymes then you can get a fairly accurate size, probably much more accurate then PFGE.
But doing the phage genome sequencing is really the best way if this is something important for you to know.
Michael J. Benedik Is there a well established protocol for it that I can follow ?
if you have enough clean DNA it’s just a set of restriction digests with good size markers on your gel.
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