how accurate Nanodrop could be? - (Aug/13/2005 )

Hi, all

we use Nanodrop to measure nucleic acid's concentration all the time. It's fast and convenient. However, the concentration varies between readings from the same sample, sometime, even big difference. I actually talked to the company selling nanodrop before, but they only questioned me if my sample was homogenized and did not give out nice solution or explanation.

anyone here has similar or better experience?

Forforfor

We've found that vortexing solutions and using 2 microliters rather than 1 improves the reproducibility dramatically. I would note that you probably have never done similar experiments
with conventional specs, just because of the pain and trouble it would entail.

I Heard that Biorad is out with a new RNA quant very much like Nanodrop, only it is reproducible and it is very informative...it even includes what type of RNA you got rather than just spec readings. I can get you a nice discount via Biorad reps, if you need to speak to a rep in US.

Second this, 2ul works well. Find it accurate.

that's quite new. We always use 1.5 ul. will try 2 ul.

I have had a few small problems, possibly caused by evaporation. It is quite dry in our labs. I never use less than 2ul either.

Measuring OD260 for RNA, I noticed that the longer the sample remained on the pedestal, the higher the reading became. One sample jumped from ~250 ng/ul to ~300 ng/ul in about 10 seconds. The estimated concentration of all samples increased quite rapidly once they were sitting on the pedestal. Even the blank increased slightly. The spectrum looked like RNA so this was probably contaminating RNA adsorbed onto the pedestal. In fact, this might be a way of checking the purity of your blank - if it increases after 30 seconds, its not pure.

The first reading I made was quite reproducible if done quickly, and luckily you can do it very quickly with the nanodrop.

thanks.

I did not read twice with the same drop of sample because it did increase because of evaporation.

and for the blank, usually I dissolved RNA in water. nuclease-free water is supposed to be very pure, isn't it ? I may need to pay attention to EB buffered sample acutually.

06:44 AM]
I have had a few small problems, possibly caused by evaporation. It is quite dry in our labs. I never use less than 2ul either.

Measuring OD260 for RNA, I noticed that the longer the sample remained on the pedestal, the higher the reading became. One sample jumped from ~250 ng/ul to ~300 ng/ul in about 10 seconds. The estimated concentration of all samples increased quite rapidly once they were sitting on the pedestal. Even the blank increased slightly. The spectrum looked like RNA so this was probably contaminating RNA adsorbed onto the pedestal. In fact, this might be a way of checking the purity of your blank - if it increases after 30 seconds, its not pure.

The first reading I made was quite reproducible if done quickly, and luckily you can do it very quickly with the nanodrop.