Dear researchers
During my thesis isolation of phage and also re-isolation from one plaque and passaging of that to reach a high concentration, I extracted the DNA and catch TEM. My TEM images show about seven types of phages that means my stock is cocktail. What can I do for the separation of different DNA in my DNA extraction, and also what Can I do for calculating the size of each phage? I appreciate your helps in advance.
Greetings,
What is the host bacteria? How many plaques were there on a double-agar overlay assay? Were they all of same morphology? If we suppose that each plaque forming unit is a single intact phage that starts multiplying on a lawn of susceptible host, you can try diluting the "coctail" extremely for further plaque purifications so that there would be just few plaques on a plate.
Some of your phages might be, as well, spontaneously induced from the host if it carries prophages.
I would first check whether anything induces from the host by UV or Mytomicin C.
Best regards,
Nick
greetings Nick
thank you for your response.the host bacteria is Proteus mirabilis and at first I saw two form of plaques (small and big) that I re-isolated one small plaque and then repeated 5 times until the same plaques were observed. but the TEM images present cocktail and multi types of phages.@Nikita Zrelovs
You must get every PFU of that phage cocktail again. Observe the morphological characteristics of the plates and I take each plate, add it to the phage buffer. Then, perform the experiment again on the agar plates to obtain PFU and the lysate from which the DNA extraction is performed.
thanks Maria Vélez
but the plaque morphology after re-isolation is the same.i did it for many times.
is it the only way that you think?
Yes, you find the same dishes. In other words, I mean that you take each phage plate individually on the double agar using a micropipette and resuspend it in the phage buffer. Then you make dilutions of each resuspended dish.
If you already did it, you should check what would be another possible problem.
On the other hand, are you using a wild or reference strain? Because when you use wild strains, phages are usually inserted into the genome (prophage), and when you isolate a new phage, the others can be reactivated to their lytic state.
Maria Vélez thanks dear for your response. yes you are right but I used ATCC strain for isolation.
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