The DyeEx procedure uses gel filtration to quickly and efficiently remove unincorporated terminators from sequencing reactions.
[A] Gel images and [B] sequence profiles of QIAprep purified plasmids sequenced using the ABI PRISM BigDye Terminator Cycle Sequencing Kit and ABI PRISM 377 DNA Sequencer. Sequencing reactions were purified using the DyeEx 96 Kit or precipitated with ethanol, as recommended by the supplier of the sequencing kit. The plasmids sequenced were plasmids from a genomic library (ethanol precipitation, gel image) or were pUC21 (DyeEx 96 Kit and ethanol precipitation, sequence profiles). Dye terminators present in ethanol-precipitated sequencing reactions cause dye blobs (indicated by arrows) resulting in unreadable sequences.
Sequence of a 4.7 kb plasmid sequenced using the ABI PRISM BigDye Terminator Sequencing Kit and purified using the DyeEx 96 Kit.
A quick centrifugation step removes storage buffer from the columns, the sequencing samples are loaded, and a second centrifugation step removes unincorporated dye terminators.