Qubit Quantification

[addyblanch]

Hi All,

I've been using Qubit dsDNA kits to quantify my DNA preps for about 12 months.

Recently we've had issues with low yields from our preps so to save Qubit reagents while troubleshooting we ran the samples on a Nanodrop. We do a phenol:chloroform:isoamyl alcohol extraction followed by ethanol precipitation and RNAse A step from bacterial culture.

With the Qubit quantification I was used to getting ~200ng/ul when we ran these on the Nanodrop the yields were coming back as 1ug/ul average! I know the nanodrop overestimates and contaminates fluoresce giving false readings but would this account for that greater difference?

I've checked the samples with a ssDNA kit also and that came back at around 40ng/ul and also ran the samples on 2 different Qubits to ensure it wasn't a fault with ours and they both read the same.

Some have mentioned the DNA might be supercoiled giving low Qubit readings but I have similar readings with fragmented DNA too.


Any ideas or thoughts much appreciated.

Adam

[SNPsaurus]

From my experience, the nanodrop usually gives a number that is 5X-10X higher than the Qubit reading. This has been consistent even when getting samples from others. The Qubit reading, in our hands, has been more useful for loading sequencers and other activities.

[TonyBrooks]

From my experience, the nanodrop usually gives a number that is 5X-10X higher than the Qubit reading. This has been consistent even when getting samples from others. The Qubit reading, in our hands, has been more useful for loading sequencers and other activities.

We see the same thing.
The Nanodrop measures everything that absorbs 260nm light and assumes it's all DNA. It's a poor assumption to make as lots of things also absorb light around the 260nm wavelength (phenol, RNA, ssDNA) etc.