Will the gel be able to separate out chromosomal/episomal/linear DNA if we load total genomic DNA (~5 micrograms) instead of lysing the cells within the wells?
Hi,
Is this similar to a comet assay?
The reasoning of lysing within the gel, is that there is no mechanical stress during lysis and DNA isolation, which enables assessment of DNA integrity far better than when using isolated DNA (during a DNA isolation there is mixing causing sheering, the DNA gets tangled, and therefore breaks, and the pipetting of course also causes DNA damage)
Because of the mechanical stress that always occurs when isolating DNA, DNA "in a tube" isn't really suitable for assays like a comet assay (and this gardella gel electrophoresis, I tried to google, but can't really find a decent reference for it), as you wouldn't be able to seperate mechanically induced fragmentation from the intrinsic fragmentation (and for example episomal fragments).
Thanks Arnoud. Yes DNA integrity is important for this assay as it attempts to separate out genomic DNA from episomal DNA (e.g. mitochondrial or viral DNA).
The reason for asking is that many times we do not have cells or tissue to work with - only the extracted DNA "in a tube"
Hi Jason,
I realize this is a problem, but not one I think you can overcome.
even if you could standardize the DNA isolation completely, doing several samples in the same batch with absolute minimal differences in mechanical stress during DNA isolation, you still would have the variable of differences between tissues (for example necrosis in the sample; and possible differences in quality of the sample due to the freezing)
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