Thursday, December 15, 2016

Negative 260/280 nanodrop reading for Nucleic Acid means what?


All of us know when we take a reading which can have minute deviations from the original value due to background noise; we do a normal thing, i.e. (S-B) , where S is the sample reading and B is the reading from the blank. Theoretically B should be 0, but as a matter of fact B shows minute absorbance due to presence of some substances those can also absorb at 260 and 280nm. Thus to get the actual amount of substance present in a given sample we subtract the Blank reading value to get the accurate sample reading. Also note that as per expectation the absorbance from the blank is always a minute value never greater than the sample value. A Nanodrop is no different. It also follows the same principle. But for a negative reading to come up, S-B has to be -Ve, which implies the B value has to be greater than S. This can happen only when the 260/280 ratio of the Blank aliquot, which I now refer as the absorbance ratio is more than that of the sample. Usually this happens if the aliquot used as Blank (control) is contaminated with nucleic acids heavily or with some other components having a higher absorbance at 260nm or if the pedestal is not properly cleaned before loading the Blank.

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