用于去除1–96个测序反应体系中未结合的染料终止剂

  • 快速的操作流程只需两步短暂离心
  • 即用型预水化凝胶过滤材料
  • 高效去除任意染料终止剂

DyeEx Kits提供离心柱或96孔板规格,应用凝胶过滤技术从测序反应体系中去除染料终止剂。将测序反应体系上样到预水化凝胶过滤材料中,短时离心后,反应体系即可用于毛细管测序仪测序。未结合的染料终止剂则会滞留在凝胶基质中。

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DyeEx 96 Kit (4)
4 DyeEx 96 Plates; 4 Collection Plates, 48-Well
63181
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DyeEx 96 Kit (24)
24 DyeEx 96 Plates; 4 Collection Plates, 48-Well
63183
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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可去除10–20 µl测序反应体系中任意类型的染料终止剂,包括BigDye、dRhodamine染料、Rhodamine、DYEnamic ET和WellRED终止剂。片段回收后,测序反应可在任意类型的毛细管测序仪上完成。推荐使用DyeEx 96 Kit按照优化的操作方法进行。信号强度高可确保较长的序列读取长度(参见"Long-read sequence")。

高品质测序

DyeEx Kits经过优化,可快速简便地去除染料终止剂,确保高品质的测序结果。与之相反,通过乙醇沉淀进行测序反应体系的回收非常耗时(参见"Comparison of DyeEx procedure and ethanol precipitation")并且效率较低。无法高效去除染料终止剂将会导致测序数据上出现染斑,使得延伸序列不可读。使用DyeEx Kits进行测序反应体系的回收,可以确保上样到测序设备的反应体系不含染料终止剂。无染斑干扰,可轻松读通序列(参见"Superior sequencing results")。 

DyeEx 流程与乙醇沉淀的比较
操作流程 DyeEx 96 乙醇沉淀
所需时间*
(12个样本,离心规格)
10分钟 >45分钟
所需时间*
(用于96个样本的微孔板规格)
N.A. >60分钟
处理 即用型离心规格 多步移液操作
序列品质 ++  +
N.A.: 不适用
* 不包括凝胶装载前干燥样本所需时间(如适用)
使用标准的实验方案.
Principle

DyeEx流程通过凝胶过滤快速高效地去除测序反应体系中未结合的染料终止剂。为了避免未结合的染料终止剂干扰测序结果的分析,因此去除染料终止剂非常重要。DyeEx凝胶过滤材料由带有均匀孔隙的球体构成,根据分子量对染料分子进行分离。当测序反应混合物上样到DyeEx柱时,染料终止剂扩散到孔隙内,并滞留在凝胶过滤材料中,而标记的DNA片段从球体间隙通过,由流出液中回收得到(参见 "DyeEx separation principle")。

Procedure

DyeEx 96 Kit用于在适配的台式离心机上使用,该离心机需配备有吊篮式水平转子,如QIAGEN 96-Well-Plate Centrifugation System。由于流程简单,因此DyeEx Kits可以较快速的除去染料终止剂(参见"DyeEx 96 procedure")。通过快速离心除去离心柱中残留的缓冲液,然后上载测序样本,接着通过第二步离心除去未结合的染料终止剂。样本此时即可直接上载到毛细测序仪上,或经干燥,再溶解后上载到测序凝胶。

Applications

DyeEx Kits可去除10–20 µl测序反应体系中任意类型的染料终止剂,包括BigDye、dRhodamine染料、Rhodamine、DYEnamic ET和WellRED终止剂。片段回收后,测序反应可在任意类型的毛细管测序仪上完成。信号强度高可确保较长的序列读取长度。

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
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For DyeEx 2.0 Spin Kit and DyeEx 96 Kit 
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