Nextera XT DNA Library Prep Kit FAQs

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  • Library Evaluation


  • The Nextera XT protocol is optimized for 1 ng of input DNA total. Illumina strongly recommends quantifying the starting genomic material. Nextera XT library prep kits use an enzymatic DNA fragmentation step and thus can be more sensitive to DNA input compared to mechanical fragmentation methods. The ultimate success of the assay strongly depends on using an accurately quantified amount of input DNA library. Therefore, the correct quantitation of the DNA library is essential.

    To obtain an accurate quantification of the DNA library, quantify the starting DNA library using a fluorometric-based method specific for duplex DNA, such as the Qubit dsDNA BR Assay system. Use 2 μl of each DNA sample with 198 μl of the Qubit working solution for sample quantification. Avoid methods that measure total nucleic acid content (e.g., nanodrop or other UV absorbance methods) because common contaminants such as ssDNA, RNA, and oligos are not substrates for the Nextera XT assay.

    Although most amplicons of interest are not likely to be high GC-content, coverage of high GC-content amplicons might have more variability compared to other amplicons.