The Nextera DNA Flex Library Prep protocol is compatible with DNA inputs ranging from 1–500 ng. For human DNA samples and other large complex genomes, the recommended DNA input is between 100–500 ng. For small genomes, the DNA input amount can be reduced to as low as 1 ng (modifying the PCR cycling conditions accordingly).
For DNA inputs between 100–500 ng, accurate quantification of the initial DNA sample is not required, and normalization of the final yield is expected.
If you are using less than 100 ng DNA input, we recommend quantification of the initial DNA sample to determine the number of PCR cycles required (see Table 1). In this case, because the final library yields are not expected to normalize, quantification and normalization of libraries before sequencing is recommended.
|Total DNA Input (ng)||Quantification of Input DNA Recommended||Recommended # of PCR Cycles||Normalized Library Yield|
When input is less than 100 ng, use a fluorometric-based method to quantify input DNA. Avoid methods that measure total nucleic acid, such as NanoDrop or other UV absorbance methods. If you use the Qubit dsDNA BR Assay Kit and/or HS Kit, use 2 μl of each DNA sample with 198 μl of the Qubit Working Solution.
UV absorbance is a common method used for assessing the quality of a DNA sample. The ratio of absorbance at 260 nm to absorbance at 280 nm provides an indication of sample purity. This protocol is optimized for DNA with absorbance ratio values of 1.8–2.0, which indicates a pure DNA sample. Target a 260/230 ratio of 2.0– 2.2. Values outside this range indicate the presence of contaminants that may cause incomplete tagmentation and adversely impact the final library yield. For a complete list of contaminants, including sources, avoidance, and effects on the library, see the Nextera XT Troubleshooting Technical Note. Dilute the starting material in nuclease-free water.