I use vectors containing whole genome of HBV for my transfections. To check viral replication, I get the supernatant of the cultured hepatocytes and concentrate the virus by centrifugation over 20% sucrose at 25,000 rpm for 4 hours.
I am wonderring if my percipitate has plasmid contamination due to cell lysis and plasmid release to the media. If so, could plasmids pass throught the sucrose along with the viruses?
With "empty" HBV capsids from E coli, they pick up nucleic acid, even without the internal binding domain. So I'd guess that yes, you would co-purify plasmids and other junk inside the capsid if not also around it.
Dialysis of the resulting HBV band *might* remove it. (although you likely have enveloped and I'm not sure then...)
You can kill the plasmids by digesting them with DNAseI. Please check the discussion: https://www.researchgate.net/post/DNAse_I_digestion_to_kill_residual_vectors
Thanks for your suggestions :)
Actually I always do DNase treatment before starting DNA extraction but something that I didn't consider was the amount of the vector which I use for transfection, which is differing from time to time while I always used the same amount of DNase!
I'm going to use enough amount of the enzyme in proper buffering condition. After DNA extraction I will check the presence of my vector by real time PCR. If there was no vector it means that DNase treatment was just fine otherwise I would think about two possibilities:
- re-considerations for DNase treatment
- As Christina pointed out there might be some free vectors swallowed by HBV capsids. In this case, for next experiment I'll take some microliter of my sample after DNase treatment but before DNA extraction (before breaking the virion to extract DNA) and also some of the extracted DNA to compare if the vector exists in both or not.
Lets see if I get any clear conclusion!
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