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How accurate is nanodropping? - (Jul/23/2013 )

Everytime I use the nanodrop for multiple readings of the same nucleic acid (pDNA) sample it gives completely different readings. The first time will read 50ng/ul. Then it will jump to 500ng/ul. Then back to a random 130ng/ul.

I mix the sample very well before nanodropping it. The sample is plasmid DNA from a Qiagen spin miniprep. This isn't the first time this has happened to me with nanodropping. I've read that it can be inaccurate, but never by that amount.

Thoughts?

-watsonkatherine-

That sort of variability is unusual, there is probably either something wrong with your prep or with the nanodrop itself. Normally it is no more or less accurate than quantitation by any spectrophotometer.

-bob1-

Do you clean the pedestal before using it (top and bottom)? I'd recommend a 1.5 or 2 ul load rather than 1 ul. Also, bubbles can cause problems, although this is rare for 1.5 ul loads.

-phage434-

I presume the different results were from different aliqouts of your sample? If so, you can pinpoint the origin of the fluctuation by measuring the same aliquot repeatedly. If the first measurement is complete, just click measure again without replacing the aliqout. If you still get large fluctuations, it's due to the machine and you should contact technical support so the machine can get some maintenance/repair. If the measurements only differ slightly, the variations you initially observed are due to a sample which is not homogenous.

I also agree with phage434 that it's better to use 1.5 or 2 µl aliqouts.

-dpo-

Base on my experience, NanoDrop is not more or less accurate than any other spectrophotometers.
It is just for making easy the process of reading of OD of more than 4 samples.
There is another machine that claims they removed the movable pedestal of NanoDrop for more accuracy.

http://www.biodrop.co.uk/

-memari-

One thing to try is to centrifuge your sample at high speed for 5-10 min before doing your readings. When you do a prep with a spin column, it is not uncommon for some of the glass fiber matrix to enter your sample - those fibres if they happen to get into your aliquot will cause wild variation in your Nanodrop reading.

-Winegarden-