DNA Input Recommendations
The Nextera XT protocol is optimized for 1 ng of input DNA. Quantify the starting material before preparing libraries.
Assess DNA purity to make sure that the initial DNA sample does not contain > 1 mM EDTA and is free of organic contaminants, such as phenol and ethanol. These substances can interfere with the Nextera XT tagmentation reaction and result in assay failure.
Input DNA Quantification
The enzymatic DNA fragmentation used for this protocol is more sensitive to DNA input compared to mechanical fragmentation. Success depends on accurate quantification of input DNA.
Use a fluorometric-based method to quantify input DNA. For example, if you use the Qubit dsDNA BR Assay system, use 2 µl of each DNA sample with 198 µl of the Qubit working solution. Avoid methods that measure total nucleic acid, such as NanoDrop or other UV absorbance methods.
Assess DNA Purity
UV absorbance is a common method used for assessing the purity 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 260/280 absorbance ratio values of 1.8–2.0, which indicates a pure DNA sample.
For a secondary indication of sample purity, use the ratio of absorbance at 260 nm to absorbance at 230 nm. Target a 260/230 ratio of 2.0–2.2. Values outside this range indicate the presence of contaminants.
Dilute the starting material in 10 mM Tris-HCl, pH 7.5–8.5. Incomplete tagmentation caused by contaminants can cause library preparation failure, poor clustering, or low quality sequencing results.
