Description
DyeEx Kits provide either spin columns or 96-well plates and use gel-filtration technology to remove dye terminators from sequencing reactions, including reactions for Sanger sequencing. Sequencing reactions are loaded onto the pre-hydrated gel-filtration material. After a short centrifugation step, the reactions are ready to be loaded onto a capillary sequencer. Unincorporated dye terminators are retained in the gel matrix.
DyeEx Kits remove any type of dye terminator from 10–20 µl sequencing reactions including BigDye, dRhodamine dye, Rhodamine, DYEnamic ET and WellRED terminators. After cleanup, the sequencing reactions can be separated on any capillary sequencer. We recommend the DyeEx 96 Kit using the optimized protocol. High-quality sequencing DyeEx Kits are optimized for fast and convenient dye-terminator removal leading to high-quality sequencing results. In contrast, sequencing-reaction cleanup by ethanol precipitation is very time consuming (see table "Comparison of DyeEx procedure and ethanol precipitation") and inefficient. The failure to remove dye terminators efficiently often leads to the appearance of dye blobs in sequencing data making stretches of sequence unreadable. Using DyeEx Kits for sequencing reaction cleanup ensures that the reactions loaded onto sequencing instruments are free of dye terminators. Without dye blobs, the sequence is easily readable throughout (see figure "Superior sequencing results"). Signal intensities are high, resulting in long read lengths (see figure "Long-read sequence"). Comparison of DyeEx procedure and ethanol precipitation Procedure DyeEx 2.0 Spin DyeEx 96 Ethanol precipitation Time required* (12 samples, spin format) 10 minutes N.A. >45 minutes Time required*† (Microplate format for 96 samples) N.A. 18 minutes >60 minutes Handling Ready-to-use spin format Ready-to-use Multiple pipetting steps Sequence quality ++ ++ + N.A.: not applicable * Does not include time required to dry down samples before gel loading (where applicable). † Using the standard protocol.
The DyeEx procedure uses gel filtration to quickly and efficiently remove unincorporated terminators from sequencing reactions. Removal of dye terminators is important to prevent the unincorporated dye terminators from interfering with analysis of sequencing results. The DyeEx gel-filtration material consists of spheres with uniform pores and separates molecules according to molecular weight. When sequencing reaction mixtures are applied to DyeEx columns, dye terminators diffuse into the pores and are retained in the gel-filtration material, while labeled DNA fragments are excluded and recovered in the flow-through (see figure "DyeEx separation principle").
Dye-terminator removal with DyeEx Kits is fast because the procedure is so simple (see flowcharts "DyeEx 2.0 Spin procedure "and ”DyeEx 96 Kit procedure”). A quick centrifugation step removes storage buffer from the columns or wells, the sequencing samples are loaded and a second centrifugation step removes unincorporated dye terminators. Samples are then ready for direct loading onto a capillary sequencer or can be dried, redissolved and loaded onto a sequencing gel.
