Monday, December 20, 2010

Quantitation of input DNA and Nextera™ fragment size distribution

When preparing Illumina-compatible Nextera libraries, it is critical to accurately quantify the input DNA amount. Variation in input DNA can affect the molecular weight (MW) distribution of libraries. The Bioanalyzer traces below show size distribution as a function of input DNA amount (human genomic DNA). As the traces show, the MW distribution increases (larger fragments) with the input DNA amount. Please note that the degree of variance will depend on a number of factors, including sample type and purity. For accurate DNA quantitation, any of the following methods can be used: qPCR, Qubit® fluorimetry, or NanoDrop™ analysis.

If a stringent size distribution is needed, there are a number of methods that can be used to size-select for the required fragment size, including AMPure® XP (for removing small fragments), Caliper LabChip® XT (for very narrow size selection), or gel extraction.

Fragment size and amount of input DNA (click to enlarge figure). Varying amounts of human genomic DNA were tagmented with Illumina-compatible Nextera™ Enzyme Mix. Tagmentation was performed using the HMW buffer, followed by nine cycles of PCR and DNA clean-up (without size selection). Red: 25 ng; Blue: 50 ng; Green: 100 ng; Aqua: 150 ng.

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