The Nextera DNA Sample Preparation Kit protocol is optimized for 50 ng of genomic DNA total. Illumina strongly recommends quantifying the starting genomic material.
Input DNA Quantitation
Quantify the starting genomic material using a fluorescence-based quantification method, such as Qubit or PicoGreen, rather than a UV-spectrometer-based method. Fluorescence-based methods, which employ a double-stranded DNA (dsDNA) specific dye, specifically and accurately quantitate dsDNA even in the presence of many common contaminants. In contrast, UV spectrometer methods based on 260 OD readings are prone to overestimating DNA concentrations due to the presence of RNA and other contaminants commonly found in gDNA preparations.
Assessing DNA Quality
Absorbance measurements at 260 nm are commonly used to quantify DNA. The ratio of absorbance at 260 nm to absorbance at 280 nm is used as an indication of sample purity. This protocol is optimized for DNA with absorbance ratio values of 1.8-2.0.