dear all,
Is anybody aware if there is a relevant differential dye intercalation between supercoiled plasmid and linear marker during Electrophoresis run? does this difference can affect DNA quantification?
Is there any reliable reference?
Thanks for any advice!
Giovanni
Since many of the DNA dyes (EtBr for one) intercalate DNA they tend to unwind DNA due to steric issues. This would suggest that indeed they will bind less to supercoiled DNA
See discussion here
http://sandwalk.blogspot.co.il/2007/07/ethidium-bromide-binds-to-dna.html
Yoram
Since many of the DNA dyes (EtBr for one) intercalate DNA they tend to unwind DNA due to steric issues. This would suggest that indeed they will bind less to supercoiled DNA
See discussion here
http://sandwalk.blogspot.co.il/2007/07/ethidium-bromide-binds-to-dna.html
Yoram
coiled. supercoiled and linear plasmid all run at different speeds . therefore size. in agarose gels due to their different shapes even without the effect of etbr.DNA generally runs 10% slower when etbr is added before running the gel due to the charge on etbr being opposite to that of the dna. small molecules will intercalate less dna because there is less room for the dye to fit into the molecule so generally all small molecules ( like primer dimer in pcr) look faint even though in molar terms there is a lot of it there so in this sense quantification is affected but usually dna quantification is done on UV spectrometry and etbr is not present so will have no effect
Supercoiled DNA binds much less Ethidium bromide (and, presumably, other intercalating agents) than relaxed DNA. This is the basis of plasmid purification using CsCl/EtBr density gradient centrifugation.
So if you are using intercalating dyes for quantification, you have to treat the relaxed and supercoiled DNA separately; they are not comparable.
If you don't know which bands are relaxed and which ones are supercoiled, this can get very complicated.
Good luck!
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