Rapid Library Library Quantitation using concentration not RFU

[GraemeFox]

Hi,
I am attempting the Library Quantitation phase of the Rapid Library Prep. set up
ie. Producing a standard curve of fluorescence through 8 serial dilutions of RL
Standard.

However the fluorometer I am using (Invitrogen Qubit 2.0) does not seem to give the option of giving results in RFU.

So....I am able to measure the concentration of my sample in ng/uL.

BUT....the 'Rapid Library Calculator' only accepts measurements in RFU.

AND...I need to ultimately calculate the concentration of my library in molecules/uL.

Is there a way to work around this that anybody knows of?

[n2onme]

Try the link below. It is used to convert ng/uL to molecules/uL with fragment size correction. You could always get a Kapa Biosystems qPCR Kit to quantitate your libraries.

http://www.uri.edu/research/gsc/resources/cndna.html

[TonyBrooks]

Doesn't the Qubit use Picogreen chemistry for fluorescence?
Therefore, fluorescence should be proportional to mass of DNA within the range of the Qubit standard curve.

I never trust the fluorescence readings for quantification as you assume all library fragments have adapters ligated at both ends. I always use the Kapa qPCR for my rapid libraries

[GraemeFox]

Thanks for the suggestions.

Having 'explored' the fluorometer a little more thoroughly I found that it does indeed give me RFU values.

This despite a phone call to Invitrogen who said that it wasn't possible on this unit!

Thanks for your help.

[madseq]

Hi,

GraemeFox, I´ve heard that the Qubit fluorometer is not able to measure RL FAM (I don´t know if this is because of wavelength or sensitivity) . Do you obtain reasonable numbers when you measure serial dilutions of the standard?

[GraemeFox]

Madseq - when performing the serial dilutions I used picogreen so I didn't have any problem.

However, my library itself was quantified using the FAM incorporated into the rapid library primers and worked fine on the Qubit.

[madseq]

Hi GraemeFox,

When you finally measured your library FAM fluorescence, did you quantify serial dilutions of the RL standard? Roche states that the Qubit is not valid for RL FAM quantification, I´ve just asked to a Roche application specialist and he says that the problem is about sensitivity.

[RCJK]

Hi GraemeFox,

I was wondering how are you able to obtain the RFU values from the Qubit? Thanks!

[GraemeFox]

Hi GraemeFox,

I was wondering how are you able to obtain the RFU values from the Qubit? Thanks!

Hi, On the 'Data' page on the unit itself you can scroll across to the right using a fairly inconspicuous scroll bar just below the rows of data.

Alternatively if you export your data via the USB port and open it up in Excel, the RFU values are also given.

Hope that helps!

[GraemeFox]

Hi GraemeFox,

When you finally measured your library FAM fluorescence, did you quantify serial dilutions of the RL standard? Roche states that the Qubit is not valid for RL FAM quantification, I´ve just asked to a Roche application specialist and he says that the problem is about sensitivity.

I didn't have a problem quantifying the FAM fluorescence of my library, or at least I didn't seem to.
Will certainly be looking into the sensitivity problem you mention though.