(while using "Nanodrop") for quantification of cDNA, one should quantify it as double-stranded DNA or single-stranded DNA?? I am using Fermentas kit for first standsynthesis of cDNA from total RNA and m not using RNaseH to degrade the RNA
ssDNA has higher optical density than dsDNA or ssRNA. See Wikipedia on Nucleic_acid_quantitation. DNA and RNA have different 260/280 absorption ratios. You could try averaging the values for DNA and RNA, but I doubt it would work.
To my knowledge, I came to know that the kit providing company suggest to use a part of your cDNA (some 5ul ) from your eluate. I tried to quantify as a ds DNA and I got huge amount as quantity but it doesnt help to synthesise my GOI. Only later I got to know this trick from my colleague and of course the company manual is saying this!
Good luck.
Because for DNA 1 OD is equal to 50ng/ul, for RNA 40ng and for ssDNA its 33ng/ul.
But UV analysis is not able to discriminate between RNA and DNA (ss or ds). You may want to use DNA-specific quantification by fluorimetry if RNA is abundant.
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