For removal of unincorporated dye terminators from 1–24 sequencing reactions
Fast procedure with only two short centrifugation steps
Ready-to-use prehydrated gel-filtration material
Efficient removal of any dye terminator
DyeEx Kits provide either spin columns or 96-well plates and use gel-filtration technology to remove dye terminators from sequencing reactions. 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.
The DyeEx 2.0 Spin Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
DyeEx 2.0 Spin procedure.
A quick centrifugation step removes storage buffer from the columns, the sequencing samples are loaded, and a second centrifugation step removes unincorporated dye terminators.
DyeEx separation principle.
The DyeEx procedure uses gel filtration to quickly and efficiently remove unincorporated terminators from sequencing reactions.
Sequence of a 4.7 kb plasmid sequenced using the ABI PRISM BigDye Terminator Sequencing Kit and purified using the DyeEx 96 Kit.
Superior sequencing results.
[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.
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.
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
DyeEx 2.0 Spin
(12 samples, spin format)
(Microplate format for 96 samples)
N.A.: not applicable
* Does not include time required to dry down samples before gel loading (where applicable). † Using the standard protocol.
The DyeEx dye-terminator removal system is designed for fast and efficient removal of unincorporated dye terminators from sequencing reactions using a simple spin column procedure. 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 flowchart "DyeEx 2.0 Spin procedure"). A quick centrifugation step removes storage buffer from the columns, 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.
After cleanup, the sequencing reactions can be separated on any capillary sequencer.
For DyeEx Kits:
Terminator removal, ABI PRISM 377, 373, 310, 3100, or 3700, MegaBACE 1000, or CEQ 2000 sequencers
For 100 x 50 µl reactions: QIAGEN OneStep RT-PCR Enzyme Mix (1 x 200 µl), 5x QIAGEN OneStep RT-PCR Buffer (1 x 1 ml), dNTP Mix (1 x 200 µl, 10 mM each), 5x Q-Solution (1 x 2 ml), RNase-Free Water (2 x 1.9 ml)