Hello,
I am transfecting linear DNA along with an Adenovirus transduction to HEK cells and need to isolate the DNA from both the linearised plasmid and viral genome at different passages for restriction digest analysis. I am not interested in the nuclear DNA
Total DNA extraction will include all genomic DNA which I fear will interfere with the restriction digest and produce a highly visible smear on the agarose gel. Ideally, I would want to just isolate cytoplasmic DNA.
Will it be okay to use the total DNA? Could I isolate extrachromosomal DNA using a standard miniprep kit, although they are meant for bacteria? Or would it be better to perform cytosolic isolation followed by DNA analysis?
Given the low quantities of linearized DNA that will be present you definitely need some form of enrichment protocol. A miniprep kit might work.
The isolation of extrachromosomal DNA, such as plasmids or viral genomes, from mammalian cells involves some unique challenges compared to bacterial DNA isolation. While traditional miniprep kits are designed for bacterial DNA extraction, they may not be suitable for isolating extrachromosomal DNA from eukaryotic cells due to differences in cell structure and the presence of nuclear DNA.
For your specific case, where you want to isolate linearized plasmid DNA and viral genomes from HEK cells without including nuclear DNA, it's important to consider the following:
Always perform pilot experiments to optimize your protocol and ensure that the DNA isolated is of high quality and purity. Consider running a gel or using a spectrophotometer to assess the purity and concentration of your isolated DNA. Additionally, you may want to perform a control experiment using total DNA to compare with the isolated extrachromosomal DNA to ensure the success of your extraction method.
I don't think you will be able to see your transfected DNA by gel electrophoresis and ethidium bromide staining unless it replicate as a virus... you will probably need to do a southern blot using different probes or maybe use PCR to detect the DNA forms you are looking for.
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