Library Quantification - Size Adjustment

[sfranzenburg]

Hi,
when you measure the concentration of your input gDNA or libraries (illumina nextera in my case), do you use the same DNA standards (=kit contents) for both? In my understanding, the standards should have a similar size than the actual sample, which is extremely different between input gDNA and 400pb libraries.
The protocol from Quantifluor mentions this problem (and recommends self-made standards), the protocols for Qubit or Quant-IT don't. Both use lambda DNA (size ~ 48,5 kb) as standards.

How do you handle that in your everyday routine?

best,
Sören

[pmiguel]

A given number of base pairs of double stranded DNA is going to bind a given amount of fluor. How would the length of the DNA molecules change the result?

--
Phillip

[sfranzenburg]

Well, that was my understanding as well.
But the fact that the Quantifluor-Manual states that you should use standards of the same size as the sample made me wonder if it is really linear.

quote from the manual:
"Even though the Lambda DNA Standard is provided with the QuantiFluor dsDNA System, we recommend preparing a standard curve using dsDNA of a similar size as the dsDNA you wish to quantitate. For example, if you are quantitating genomic DNA, you should prepare a standard curve using a genomic DNA sample of known concentration."

I just wondered if people actually do that? But if, how do you get a sample of known concentration to calibrate your method that measures your concentration. Chicken-Egg problem...