Hi,

you will always get differences among the two quantification tecniques.

It is due to the adsorbance of nucleic acids. Indeed, nucleic acids absorb UV light at 260 nm due to the aromatic base moieties within their structure. Purines (thymine, cytosine and uracil) and pyrimidines (adenine and guanine) both have peak absorbances at 260 nm.

The problem is that actually not only  dsDNA adsorb at 260 nm but also all nucleic acids and polyphenolics compounds.

:-)

Hi Amit,

DNA quantification using gel electrophoresis is much more a semi-quantitative method used for a ''quick and dirty'' readout that let you know that you how much DNA there is in your sample in comparison to the ladder intensity. Results will vary depending on ladder preparation, concentration of dye, background in the gel, etc.

DNA quantification using a nano-drop is much more quantitative and reproducible as long as you stay in the recommended dynamic range of 2ng to 3700ng. Results should be robust as long as you have a good blank.

So, you should expect a difference between the two methods as they are not ment for the same usage and interpretation. 

Best,

Nicolas

Another reason is that the nanodrop will detect ALL nucleotides including primer dimers, unused dNTPs, etc and not just your desired product.  As Nicolas said above, gel electrophoresis is semi-quantitative and is good enough to give a rough estimate.  Which method to use really depends on how accurate you need to be with the next step in your experiment.  FYI, the nanodrop will also give a false signal from many reagents/contaminants such as DMSO, RNA, proteins, etc.  If your DNA is not very clean, then I'd rely more on the gel electrophoresis.

For quantification I would use a decent method like the Qubit (although it only has two controls to set a calibration curve) or AccuClear (a bit more time consuming, but more accurate).

See also this publication:

Article Fluorescent cyanine dyes for the quantification of low amoun...

Dear Friends,

Thanks for your replies.

Let me explain my query in more detail.

By analysing a ladder standard on Micro spectrometer I am getting answer 503ng/ul, which is quite correct, the standard is of 500ng/ul. Similarly, I analysed extracted sample DNA the reading showed, 3280 ng/ul.

Same samples were analysed on gel electrophoresis. Considering the ladder concentration and its band intensity obtained on gel electrophoresis, comparing it with the samples intensity results in 1809 ng/ul concentration, which is almost half of the value obtained by micro spectrometer .

I am not able to understand how two techniques are giving two different results for the same sample, viz : 3280 and 1809.

please help.