For removal of unincorporated dye terminators from 1–96 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.
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Product Cat. no. List price:
DyeEx 96 Kit (4)
4 DyeEx 96 Plates; 4 Collection Plates, 48-Well
63181
$852.00
DyeEx 96 Kit (24)
24 DyeEx 96 Plates; 4 Collection Plates, 48-Well
63183
$4,515.00
The DyeEx 96 Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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DyeEx separation principle.|DyeEx 96 procedure.|Long-read sequence.|Superior sequencing results.|
The DyeEx procedure uses gel filtration to quickly and efficiently remove unincorporated terminators from sequencing reactions.|A quick centrifugation step removes storage buffer from the wells, the sequencing samples are loaded, and a second centrifugation step removes unincorporated dye terminators.
|Sequence of a 4.7 kb plasmid sequenced using the ABI PRISM BigDye Terminator Sequencing Kit and purified using the DyeEx 96 Kit.|[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.|
Performance

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. Signal intensities are high, resulting in long read lengths (see figure "Long-read sequence").

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"). 

Comparison of DyeEx procedure and ethanol precipitation
Procedure DyeEx 96 Ethanol
precipitation
Time required*
(12 samples, spin format)
10 minutes >45 minutes
Time required*
(Microplate format for 96 samples)
N.A. >60 minutes
Handling Ready-to-use
spin format
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.
Principle

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").

Procedure

The DyeEx 96 Kit is used with a suitable table-top centrifuge equipped with a rotor with swing-out buckets, such as the QIAGEN 96-Well-Plate Centrifugation System. Dye-terminator removal with DyeEx Kits is fast because the procedure is so simple (see flowchart "DyeEx 96 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.

Applications

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. Signal intensities are high, resulting in long read lengths.

Feature
Specifications
Binding capacity 10–20 µl
Elution volume 10–20 µl
For DyeEx Kits: Terminator removal, ABI PRISM 377, 373, 310, 3100, or 3700, MegaBACE 1000, or CEQ 2000 sequencers
Format 96-well plate
Processing Manual
Removal <10mers 17–40mers dye terminator proteins Dye terminator
Sample type: applications Sequencing reactions
Technology Gel filtration

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Kit Handbooks
2
References
0