The lysogenic phage genome, which I isolated by the traditional phenol-chloroform method, appears small on 0.6% agarose gel electrophoresis. When I compare it with Marker, it is equivalent to 250 bp. Is it because the genome is degraded or somehow broken? Does being a lysogenic phage affect this? I'm looking forward to your views.
I don't think you mean 250kb because that would be very very large and it would not migrate in a 0.6% gel. Do you mean 2.5kb or 250bp, or something different?
Regardless, if this is too small a band, how do you know that it really is the phage and not something else? Maybe you just aren't seeing the phage DNA?
Thank you for your answer. It's 250 bp, sorry i fixed it. I'm sure it's phage. I isolated genome of from phage filtrate that i did a lot of experiments (spot tests, MOI, host range, adsorption range) with it. But I'm stuck at the genome isolation part. I don't think I can get a whole genome when I isolate with kit or phenol-chloroform. Do you have any suggestions for this Sir ?
Clearly you are not going to have a phage genome of 250bp, but I’m not sure whether this is an excision product of some form or a plasmid. You could do a mini prep of the host cells and see if you get it as well.
it might be that you just don’t have enough phage to start with in order to get enough DNA to see. What titer of phage did you start with before making DNA?
It is possible that the small band size you observed on the gel is due to DNA degradation or fragmentation during the isolation process. This can happen if the DNA is exposed to harsh conditions, such as prolonged exposure to high temperature, or mechanical shearing during extraction or handling. Lysogenic phages are not inherently more prone to DNA degradation, but the specific isolation method you used may have contributed to the observed band size.
To confirm whether the DNA is intact or fragmented, you may want to perform a gel electrophoresis analysis of the extracted DNA before proceeding with any downstream applications. Alternatively, you could also quantify the DNA using a fluorometer or spectrophotometer to determine the concentration and quality of the extracted DNA. If the DNA is indeed degraded, you may need to optimize your isolation method to minimize DNA damage or consider alternative methods for extracting the phage genome.
Here are some literature and scientific papers that may be useful:
These papers discuss various methods for isolating phage DNA and optimizing DNA extraction, which may be helpful in improving your isolation method and ensuring the quality of the extracted DNA.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
Is it possible to reach out which phage is lytic or which one is lysogenic from their genome? and what's the computational and experimental analysis that we can separate them from each other?
If you have the sequence of the entire phage genome, it might be possible if it includes genes that show homology to known lysogenic phage elements, such as a repressor gene or site specific recombination genes for integration. This wouldn't prove a phage is lysgonic, but would strongly suggest it might be.
Did you ever figure out what the 250 bp band was?
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