Thanks for the reply Michael. The size issue is really interesting - do you know the size of the fragments in the standards that come with the kits? In addition, it seems you are still referring to the dsDNA kit. have you used the ssDNA kit? Thanks.

We tried to use the Qubit ssDNA kit to quantify samples from a low yield extraction. The ssDNA kit is not very sensitive to lower quantities of DNA. So if you sample has low concentrations the Qubit has a hard time detecting the sample.

We have also compared the Qubit to a spectro results, and the Qubit consistently measured higher concentrations and was more consistent than the spectro (for those samples that we could measure). Several in the labgroup have used Qubit for next-gen quantification of DNA (ds) with the standards that come with the Qubit, and swear by the thing as giving them a great starting point for gel quantification and additional steps from there, but I can't speak to that. Nor does that really help with ssDNA as you can't gel quantify it.

We are denaturing dsDNA and therefore have expectations of what the quantity of ssDNA should be (we quantify the dsDNA using a dsDNA qubit kit). We have basically given up using the ssDNA kit. It does not seem to measure ssDNA. We have obtained a new kit free from LifeTech and got the same results. Now after a bunch of tests using PCR products and genomic extraction we are using the dsDNA HS kit (that seems indeed to work fine), assume some degradation after denaturation (based on our experience) and move on. There does not seem to be that many people using the ssDNA qubit kit. Thanks for replying to my question.

Hi,

Like the others we have tried to use the Qubit ssDNA kit without results we are happy with. The sensitivity is low and the base line measurements drift rapidly with time after addition of the dye to the DNA. While it is a nice sounding technique I would avoid using the Qubi for ssDNA measurementst. However if you had the time adapting the technique to a plate based format would likely help - you would need to look at a range of dyes aside from those in the Qubit kit (picogreen I think). I have dug up an interesting (though dense) photochemical paper discussing some of the issues we and others have been seeing with these dye binding assays (Photochemistry and Photobiology, 2001, 73(6): 585–599

Photophysical Properties of Fluorescent DNA-dyes Bound to Single- and Double-stranded DNA in Aqueous Buffered Solution G. Cosa, K.-S. Focsaneanu, J. R. N. McLean, J. P. McNamee and J. C. Scaiano). The decay issues appear to be an issue with whichever dye system is being used. So where to from here..... The DsDNA kits will work well due to the stability of the dye-DNA complexes which also appear to be less prone to photochemical degradation. The detection of ssDNA is a lot more problematic and using a proxy as has been mentioned above and has been 'calibrated' for the use intended is likely to be the best approach.

One final thing is that in our hands the Qubit is fine for both DNA and RNA quantification. Differential results between the Qubit and spec (nonodrop or similar) indicates there are contaminents in the sample. When we get good correlation between the 2 methods we know we have very good quality RNA/DNA. Consequently as part of our QC for samples moving forward to qPCR or NGen sequencing we do both analyses.

Like Michael Derr, I also had a bad experience with Qubit.

On comparing its reading to Nanodrop and to gel also, I felt that Qubit gave lesser readings that what is actually.

Hi,

We had good results with Qubit for dsDNA (BR reagent). This was confirmed by measuring the concentration of standards (i.e. DNA ladders) commercially available (they were consistent with declared concentrations and UV readings). It looks that the dsDNA purified by ethanol precipitation gives readings very similar in both by UV absorbance measurements and Qubit (BR).

If the DNA comes from minipreps or spin column purifications UV readings is likely affected by contaminants in spin column buffers (these buffers all absorb highly in the UV region normally), while Qubit gives good results.

We measured the concentration of a dsDNA ladder by UV before and after a spin column purification, and after purification (resuspending in the same initial volume) the concentration appeared much higher than the original one!?

We had bad results with HS reagent.

ssDNA concentration would appear quite difficult to determine as one would expect that the ssDNA structure is highly variable in terms of hairpins and region partly double stranded ("heterodimers") (random occurrence of short complementary regions).

Even a simple reading at UV of pure ssDNA in pure water would be possibly significantly sequence-dependent and sensitive to the number of dsDNA regions formed (which absorb notoriously differently) but I would suggest it could be the most reliable.

Uvette (eppendorf) are disposable cuvettes which could be use in UV readings by spectrophotometers with as little as 70ul and I would reccommend them for ssDNA.

I did not try Qubit for ssDNA but it is not clear to me what is the principle used as the average structure of ssDNA is likely extremely variable and sequence-dependent.

Ideally one should measure the UV absorbance at 95C to be sure no secondary structures are formed but this is clearly not possible at the bench level.

I also do not know which is the most convenient way to mesure ssDNA concentration honestly but Qubit is really good for ds

Max, wonderful answer. That males a lot of sense. Yes, we use the Qubit for dsDNA routinely and it works great. But the ssDNA system in our hands does not work.

Dear Dina,

Thanks for your comments.

I actually have the same doubts about the ssDNA concentration methods and I think I will just go with UV reading in my case but I will keep an eye at any other suggestion on this discussion anyway.

Thanks again,

Max.

 I have also faced similar problems of the Qubit measurement of ss DNA. There is a much variation in the Qubit results of the same sample if you run samples within 2-3 minutes intervals. Do you have any recommendation how to solve this issue?

During a recent project, I discovered that the proprietary SYBR gold II dye that is in Invitrogen’s EGel EX Agarose gels carries through gel extraction and purification and interferes with the ssDNA Qubit quantitation. It even somehow carries through when there is no DNA present. Try it for yourself. Crack open a gel, slice out a piece and purify it. You will get a significant Qubit measurement in ng/ul, which will change depending on your elution volume.

The dye generally increases the concentration measurements, but to what degree is dependent on the amount of DNA present. The lower the amount of DNA present, the more pronounced the effect of the dye. The more sample you add to the Qubit assay (2ul, 5ul, 10ul) there is a general trend in my hands of measurements going down, which I assume is due to some level of competition between the gel dye and the qubit assay dye. Ethanol precipitation does not remove the dye and tech support was not able to provide me with any alternative solutions at this time. They did not seem aware of the problem at all, so hopefully they are working on a solution to make it possible to accurately quantify gel-purified DNA on the Qubit.

I am facing the same problem with measuring ssDNA. Just found out it's better to wait a longer time than 2-3 minutes (in my case 10-15 minutes) after adding the sample to the buffer and vortexing.

This is even true for the standard #2 provided along the kit.

Has anyone used Qubit dsDNA kit for cDNA?