I am isolating mycoplasma phages by "picking" an isolated plaque and then culturing in Mycoplasma broth. After eluting the viral nucleic RNA/DNA and running the enzyme digest
there are no bands detected on the DNA gel. I am using EcoRI. Also the 260/280 ratios are high,
around 3.0 to 3.3.
Hello Larissa,
If the A260/A280 ratio is 3.3, what you are looking at is probably not nucleic acid, which explains why you don't see any band on the DNA gel. Another problem could arise if your phage is a single-strand DNA phage. First, it won't be cut by restriction enzymes, and second, signle-strand DNA stains poorly with intercalating dyes such as ethidium bromide compared to double-strand DNA. Another problem may be the titer of the phage stock that you start from for extracting the DNA. For a 50 kb phage (e.g. lambda), it takes 2 x 10exp10 phages fore a theoretical yield of 1 µg of dsDNA.
Pierre is correct, "Pure" DNA should be at about 1.8, while RNA should be around 2.0. pH can have some effects, but not enough to change it that much. You may find this useful. https://www.bio.davidson.edu/projects/gcat/protocols/NanoDrop_tip.pdf
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