Microvolume UV Spectrophotometry for Nucleic Acid Quantification: The new (good) way

Actually it was not that “new” since this method founded in the year 2000. At least the founding of this method assign new era of nucleic acid quantification. If I summarized of problems caused by old way nucleic acid quantification, there are two big problems, first it needs relatively big volume of sample in order to fit in cuvette volume, meanwhile sample can be very rare to get. Second problem is dilution. Dilution is tedious. Third is cuvette cleaning is time consuming. Fourth is calculation can be complicated.

Nucleic Acid Quantification: The new (good) way

In micro volume UV spectrophotometry, all we have to do is put the sample in micro volume (1-2 microlitre) into a place called pedestal. Since this pedestal made of stainless steel conditioned hydrophobic, the liquid will form nice shaped spherical “ball”. Next step is pulling down so called “arm“. The low end of arm will connected with pedestal through micro volume liquid forming column. The light will be transmitted from pedestal to the arm through this liquid column. The maximum distance is 1 millimeter.

Absorbance can be calculated from light transmitted and light detected in few seconds. All standard parameter (nucleic concentration, purity ratio) automatically calculated using software also in few seconds. The software also shows a graphic of full range measurement of absorbance from wavelength 190 to 340 nm. With this method, all problems in old method were solved, and even more, characteristic of good purified nucleic acid can be known from the graphic.

From one sample to another, all we have to do is wipe the pedestal using appropriate laboratory tissue paper. It is so simple and not time consuming. If you do concern of sample to sample contamination, it is statistically insignificant. Just give extra ddH2O between sample measurements to make you feel comfortable, even it is not mandatory and it will slow you down.

Everything is done in only 3-5 seconds! Just with putting your sample in very low volume (1-2 microliters) into a pedestal, pull down the pedestal arm and one click on measure button gives you all the data you need in quantification within 5 seconds.

Not forget to mention, blank is mandatory as this is the same spectrophotometry. Before we give the blank, measure button will not appear. Blank is the same thing we measure except it does not contain the sample. Meaning blank is the solvent of the nucleic acid. Give the relevant buffer will be ok.

Micro volume UV spectrophotometry is very useful due to a very demanding condition of current research situation such as high volume of sample in which tedious method will suffer the researcher with non research objective related problem. Of course some price should be expensed for such technology; the price of instrument can be two or third time regular spectrophotometer. Anyway, I’m sure it will be worth since there is no need to spend any cost (example consumables) when running the instrument except monthly pedestal conditioning chemical and annual calibration which requires a simple reagent.

Once I have experience. At that time, it seems the nucleic acid extraction is failed for no white haze at all seen on the final tube. Luckily I bring along this instrument, and then I took a bit of the sample to find out. I got 5 nanogram of DNA which actually still possible to do a Polymerase Chain Reaction (PCR), so, the nucleic acid extraction is not failed.

I also know that one of national institution in Indonesia is using real time PCR for quantifying nucleic acid. It was using the same micro volume of sample but it does not count the time and reagent cost yet. Can you imagine how long the time needed for this real time PCR technique? It will take about 1.5 hour process. It becomes 5400 seconds process against 5 seconds process. Not to mention the cost of reagent needed which will cost about USD 40 per sample meanwhile micro volume UV spectrophotometry does not cost you at all.

Micro volume UV spectrophotometry used in recent biology molecular technique such as microarray and next generation sequencing. Concentration and purity of nucleic acid as input in certain technique becomes critical in order to get reliable outcome/result.

I still remember attending a class when I’m in college; the lecturer lectures Lambert Beer Law. It was amazing for me to see that the lecture is useful.
Bynix Saya hanyalah blogger pemula yang ingin sukses didunia blogging

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