Qubit - wrong values due to GFP?

[AnneH]

Hi,

I performed a Qubit measurement on genomic DNA (BR) and took 2.5 µg according to the measurement for a reaction with a GFP-tagged protein.
Afterwards I did another measurement (HS) (because I thought due to clean-up steps I would probably have less DNA), but weirdly now I have about double the amount of DNA in there.

I also measured the original DNA again with HS (comes out at about the same amount as BR).

Now I was wondering if maybe residual GFP-tagged protein could influence the measurment? Does anybody here know anything about that? Or maybe experienced something similar?

Thanks in advance! Anne

[cliffbeall]

I am pretty sure it will interfere, the Qubit fluorophore is green (I believe it's picogreen?). You might be able to measure the sample without adding the fluorophore and subtract that out as background?

That being said, I wouldn't expect GFP to survive a typical DNA purification -they usually involve proteases and denaturants.

[bombardior]

that qubit reagent is orange in color, which, coincidentally, is the same color as sybr green, so i think gfp may indeed interfere with qubit readings.

[nucacidhunter]

Now I was wondering if maybe residual GFP-tagged protein could influence the measurment? Does anybody here know anything about that?

Loss of DNA during purification is expected. Increase in measured DNA amount after clean up is most likely false. It could be due to contaminants (salts, proteins and other substances) that has not been completely removed. There is a list of common contaminants and their effect on quantification with PicoGreen reagent in the product manual: http://tools.lifetechnologies.com/co...ls/mp07581.pdf

[AnneH]

I guess the next time I use this GFP-tagged protein, I will set some of it aside and do an extra Qutbit measurement with just this protein and without DNA.

Thanks nucacidhunter for the Link, unfortunately the only thing from that list that might still be in there could be sodium chloride, and this would lead to a decrease, not an increase :-/

[nucacidhunter]

I do not know details of your protocol, but PicoGreen normally is excited at 480 nm and fluorescence emission is collected at 520 nm. GFP unlike PicoGreen is a protein and has to be in soluble form to be active and fluoresce under correct excitation condition. You may check fluorescence spectral viewer (http://www.lifetechnologies.com/au/e...#product=P7589) to see if your GFP type can be exited and emit at the same wavelength as PicoGreen.

[bilyl]

Hi,

I performed a Qubit measurement on genomic DNA (BR) and took 2.5 µg according to the measurement for a reaction with a GFP-tagged protein.
Afterwards I did another measurement (HS) (because I thought due to clean-up steps I would probably have less DNA), but weirdly now I have about double the amount of DNA in there.

I also measured the original DNA again with HS (comes out at about the same amount as BR).

Now I was wondering if maybe residual GFP-tagged protein could influence the measurment? Does anybody here know anything about that? Or maybe experienced something similar?

Thanks in advance! Anne

If you are really concerned, try spiking in a microliter of Proteinase K into your Qubit tube and see if that changes the signal. Try it on some "no-GFP" DNA to have your negative control.

I wouldn't put the ProK into your main DNA stock, as it might interfere with downstream assays unless you clean it up really well.

[IdahoRAD]

So, you are making sure to only consider the amount of DNA, and not the concentration? Changes in elution volumes could make it "look" like the DNA content is increasing.

[kerplunk412]

Treating some of your sample with ProtK sounds like a good idea to me, but you might want to do that in a separate tube and then just use an aliquot of that reaction for Qubit. Although just doing it in the same tube would probably work as well.

Also, have you tried just using the Nanodrop? It sounds like your DNA may be concentrated enough that old fashioned spectroscopy should give you a fairly accurate reading.

[AnneH]

So, unfortunately I don't have anything left of that sample and I treated it with Proteinase K before the measurement.
@IdahoRAD: Yes of course I consider the amount. I know that I went into the reaction with about 2.5 µg and came out with about 5 µg.

I also measured the DNA with Nanodrop, and here I measure an even higher increase. Unfortunately i didn't write down 260/280 nm ratio :-/

[AnneH]

I performed a measurement, with different input of the GFP-tagged protein (without DNA) and I can in fact measure "dsDNA" with the High Sensitivity kit.

It wasn't high enough though to explain my miraculous increase in DNA in this sample completly though.
But well at least one question answered: GFP can influence DNA measurement with Qubit.