Microvolume UV Spectrophotometry for Nucleic Acid Quantification: The old (not good) way

Nucleic Acid Technique is my favourite subject when I’m in college. But one thing that makes me discomfort is measuring DNA concentration. In old (not good) way, we have to dilute our DNA obtained from extraction into bigger volume. The reason we have to do so is volume of DNA we get from extraction is very less than the volume of cuvette in conventional UV spectrophotometer. Usually we dilute DNA into 40-50 fold dilution. After that we measure the absorbance of diluted DNA in several UV wavelengths like 230, 260 and 280 nm. Not forget to mention, we also measure the blank absorbance for correction. With specific coefficient of nucleic acid 50 ug/ml correlate with 1 absorbance, we’re ready to calculate the DNA concentration.

The old (not good) way

Based on Lambert-Beer’s Law, A = E. d. c, where A is absorbance, E specific coefficient, d is path length of array in spectrophotometer and c is concentration. All data acquired is deducted by blank absorbance, then net Absorbance in 260 nm multiplied against 50 ug/ml. We haven’t got the DNA concentration yet before put dilution factor into calculation. After dilution factor calculated, finally we get the DNA concentration. Remember, the step of DNA concentration measurement using this old (not good) way start from diluting our samples, not forgetting the label what cuvette contains what sample. Dilution factor also must be kept safe since very critical to final result. Diluting means lots of pipetting, drying out your consumables (pipette tips), it also needs lots of microvolume tubes. One important thing also consumed is time (and energy), whew…

After DNA concentration obtained, we also have to calculate some parameter, first 260/230 nm as purity of DNA/Nucleic Acid against Phenol and 260/280 nm as purity of DNA/Nucleic Acid against Protein. Good quality DNA has value of 260/230 at 1,8 and 260/280 nm at 2,0. That is the ideal condition.

Regarding DNA amount measurement/quantification above, can you see it takes us too much resource and energy, can you imagine if your sample is quite a lot, let’s say 50 samples/day, measuring DNA concentration with old (not good) way can take half of your day. The same protocol also can be applied to RNA as they consist of the same nucleic acid with DNA.

Thank God there are creative people in this world catch the pain suffered by researcher in quantifying DNA/nucleic acid (to be continued soon)
Bynix Saya hanyalah blogger pemula yang ingin sukses didunia blogging

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1 Response to "Microvolume UV Spectrophotometry for Nucleic Acid Quantification: The old (not good) way"

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