Nextera XT input considerations
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Sample input is the most important factor in a successful Nextera XT library prep.
The first step in the Nextera XT library preparation workflow is the tagmentation reaction, which involves the transposome cleaving and tagging the double-stranded DNA with a universal overhang. The tagmentation efficiency is a key factor for the success of the library preparation.
The Nextera XT library preparation kit is optimized for 1 ng double-stranded genomic DNA.
Quantify the input DNA with a fluorometric method, such as PicoGreen or Qubit. Avoid using methods that quantify total nucleic acid, like UV absorbance methods.
The transposomes are end-point enzymes, in that they cleave double-stranded DNA one time only and then the step is complete. Therefore, successful tagmentation is highly dependent on the input mass.
Using > 1 ng can lead to undertagmentation of the sample.
Using < 1 ng can lead to overtagmentation of the sample.
For any feedback or questions regarding this article (Illumina Knowledge Article #1023), contact Illumina Technical Support .