T
he success of the library preparation step greatly affects the outcomes of next generation sequencing (NGS) experiments. After researchers isolate nucleic acids, they must perform careful quality control (QC) tests on the resulting material before proceeding with library preparation. Assessing nucleic acid concentration and sample purity is best conducted by two different instruments: a spectrophotometer and a fluorometer.
Spectrophotometers quickly measure the absorbance of a sample using ultraviolet-visible (UV-Vis) spectrophotometry. This value at a species’ peak absorbance wavelength is proportional to the concentration of that species in the sample. Traditional spectrophotometers use transparent cuvettes to hold liquid samples, but newer microvolume models measure extremely small volumes (0.5-1 µL) on a stage. While spectrophotometers quickly measure absorbance and calculate concentration, they are not very specific for nucleic acid quantification. In the case of DNA library prep, dsDNA, ssDNA, RNA, and free nucleotides absorb at the same peak wavelength of 260 ...
