We have isolated Phage DNA with the Qaigen Phage DNA Isolation Kit. Those samples were treated multiple times with various enzymes. We have done single, double, and triple digests to no avail. We have tried using different buffers (made by us and purchased) and still have no banding patterns on our sample DNA. We have been using lambda phage DNA as our control and it has performed as expected, with banding and minimal smearing. We wonder if maybe there is methylation that is inhibiting the enzymes, or if perhaps the sample DNA needs to be re-isolated. I wonder if anyone has had similar problems and if you have encountered any solutions.
As suggeste by Azhar, methylation could be an explanation. However, it is strange that none of the restriction enzymes you tried seems to work, since most modification methylases are site-specific, thus you would expect that at least some restriction enzymes would be able to find unprotected sites. Another possibility is that your DNA prep contains some inhibitor(s) that prevents efficient digestion. One way to check for this would be to spike the digestion mix with some lambda phage DNA and see whether it is still digested in the presence of your DNA prep.
Have you got sequence of the phage DNA. If yes then do restriction analysis first and them aim to perform sequential digestion with different restriction enzymes.
I have to agree with Pierre Béguin that I don't think methylation is the explanation. You need site specific methylation patterns to inhibit restriction digests and the likelehood that you are methylating at all of the sites for different enzymes is very unlikely.
Do you know anything about the phages you are isolating? Some bacteriophages are naturally resistant to restriction, for example phage T4 has modified hydroxymethylcytosine bases in its DNA that makes it resistant to restriction digestion. A number of other phages also use different modified bases.
Is this one specific phage that is giving you problems or a number of different ones? If you are having problems with just one, this could be the answer. But it is worth doing the mixing experiment that Pierre Béguin suggests where you mix your DNA and lambda DNA to see if there is something inhibitory in your phage DNA prep that is blocking digestion of lambda DNA as well.
What is the host bacterium for your phage? Perhaps knowing that might give a hint.
If you have a TEM image, then make sure that your phage comes under the class of dsDNA phages.
Michael J. Benedik Pierre Béguin we are using 16 different host bacteria and all of the isolated phages for all of those bacteria are resistant to digestion (so far). For example, a few of the host bacteria we have phages for include: S. flexneri, K. aerogenes, S. sonnei, C. freundii, etc. It does seem that the problem lies in the DNA prep. We are using the elution buffer given by the isolation kit and have performed three washes on the samples.
I agree that it must be something in the conditions of the samples. First you might want to try a reconstruction experiment, add some lambda DNA to your normal amount of some of the phage DNAs and see if the lambda still digests or not. That would tell you if you have an inhibitor. But you might just try to do an ethanol precipitation of your phage DNA, that is usually the best way to clean it up (or a spin column).
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