Hi everyone. I have cryo EM sample containing DNA-bacteriophage. The bacteriophage is purified in CsCl gradient and concentrated in a centricon concentrator with 100 kDa cutoff. The problem is that I can see high DNA contamination in the background. If a DNAse is added to my sample all bacteriophage particles are gone. Therefore, I need to purify it some other way. Any ideas?
could you restriction digest with 2 or 3 enzymes which do not have cut sites in your phage and then run the DNA on a low percentage agarose gel and excise the band of the right size and purify?
Thanks so much for your reply Paul. You probably misunderstood my question (or maybe the question is not clear). The problem is that I need to purify whole bacteriophage particles containing DNA ("full particles") without any "exterior" DNA floating in the sample (or at least with short DNA chains which are not visible in cryo-electron microscope).
Usually, tailed dsDNA bacteriophages are quite resistant to DNAse treatment. We use benzonase pretty routinely. Maybe your DNAse is contaminated with proteases? otherwise you would probably have to prep on a CsCl gradient again and image immediately afterwards, before the particles have a chance to eject more DNA (if that is the source of your DNA contamination).
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