I found a kind of DNA virus in my insect sample soaked in pure ethanol. I got a sequence from the virus. I want to purify the virus particle or the DNA of this virus to sequence the genome of this virus. I do not know if I can get the viral genome without the insect genome contamination successfully. Can anyone give me some advice?
Ethanol won't damage the DNA. this is important that you know where it is replicating. You need to concentrate your sample via centrifuge. now, the DNA you want is in the membrane/capsid/ envelope.. I haven't tried this personally.. I'm sure there is a kit you can buy. But I'd prob lyse cells.. Add Dpn1 to digest all methylated genomic DNA then heat inactivate. Then I'd use TRIzol to break open the viral packaging then do chlorophorm/ethanol precipitation. and from there, I would probably use RACE
Not virologists, obviously...B-) You can actually use ethanol to precipitate stable (non-enveloped) virions, AND get infectious virus back later, so that is not necessarily a problem.
You can purify the virus from insects using very standard protocols - given that insect viruses often reach very high titres, squashing and thoroughly homogenising a bunch of bugs in some ordinary buffer (eg: PBS / phosphate pH 7), centrifuging at say 15K rpm for 10-20 min to pellet garbage, then putting the supernatant onto a sucrose / iodixanol density gradient ad ultracentrifuging it. Small isometric viruses will take a good couple of hours to sediment to a position you can suck off the band; big viruses will obviously go faster.
THEN do DNA extraction!
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