I am Concepcion Calvo researcher at the University of Granada. We are studying the microbial diversity in the composting process of sewage sludge. So we're doing the isolation of DNA of isolated cultures for subsequent sequencing of the 16S RNA. Also, we are doing DNA extractions from samples of compost for subsequent pyrosequencing.
In our lab we use two techniques: Qubit and nanodrops to know the quality and concentration of DNA isolated. But when we carry out these determinations with both methods, the results are quite different. I wonder if anyone has experience comparing these two techniques to determine concentration and purity of DNA. And which of these procedures would be the most reliable test?
I have used both of these systems for evaluating nucleic acid concentration. The Qubit is certainly the more accurate method of determining concentration and so we use this for our NGS library quantification and other concentration critical applications.
The Nanodrop is not as accurate- we generally us this for measuring quantity/quality of nucleic acid when we expect concentrations 20 ng or more, and do not require high accuracy. We also find it useful for giving us an idea of how to dilute a sample if we want to run it on the Bioanalyzer or a Flash gel.
I haven't directly compared purity measurements using the two systems, but would think that the accuracy would follow that of the concentration determination (i.e. the Qubit would be the most reliable). However, as it is necessary to add the sample to each of 2 different assays, then this may be a source of increased uncertainty of the measurement, compared to the Nanodrop which takes the purity measurement from the a single sample addition.
I suscribe the answer of Gerwyn Jones.
Just to add couple of things:
1) "Qubit" measures total quantity of nucleic acid, whereas "Nanodrop" measures concentration. Accuracy is much higher for Qubit (using the adequate kit for a certain range of concentration, of course), but, as Gerwyn wrote, pippeting error must be taking into account, not happening in nanodrop.
2) I have experienced problems with Qubit when the RNA sample was coloured (sometimes it happens when RNA extraction using trizol is made starting with a big amount of grounded olive leaves). In this case, concentration measurements with Qubit were inconsistent.
Hope it helps!
Qubit and Nanodrop are the best, we use both. Qubit readings are sometimes lower- esp when DNA preps are "dirtier", as Qubit assays are very specific towards analyte. Nanodrop is great because its faster, cheaper, and also provides spectrum- so you can check 260/280 and 260/230 etc which is extremely important
Well I have some experience with Nanodrop and I think it is not a robust and consistent method. The results obtained through this technology are very variable. Qubit is much better for determining DNA concentration.
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