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Sequencing reaction purification (SRP) is typically performed using gel filtration, solid magnetic substrate, membrane platform, or standard ethanol precipitation. The samples are centrifuged, magnetically separated, vacuum filtered, or precipitated and centrifuged, respectively, in order to eliminate salts, unreacted dNTPs, and dye terminators from the extension products. We have developed a surface-modified microplate to perform SRP without the need for these manipulations.
With this technology, the extension products are selectively adsorbed to the surface-modified multi-well plate, washed with alcohol, and eluted in water. The system was evaluated in 96- and 384-well plate configurations on an ABI 3730XL DNA Analyzer using the BigDye v3.1 chemistry. Results were obtained with typical Phred20 scores of 800.
The plates performed reproducibly over a wide range of reaction conditions. The system was tested using sequencing reaction volumes ranging from 5 to 10 μL, DNA template masses ranging from 10 to 100 ng, and BigDye volumes ranging from 125 nL to 2 μL. Results were obtained both manually and via automation.
With no adjustment to the protocol, the modified plate system was very robust to variations in template and BigDye concentration as well as sample volume. Using a Biomek FX workstation, plate processing times were less than 30 min. Thus, this new technology offers a robust, high-capacity, and fully automatable dye terminator removal platform to work efficiently with reduced volumes of dye terminators.