Excess unincorporated, non radioactive labels can cause high background fluorescence in automated sequencing gels. For optimal sequencing results, remaining labeled dideoxynucleotides should be removed prior to electrophoresis. The Ultra-Sep™ Dye Terminator Removal Kit is designed for effective and reliable removal of unincorporated terminators from sequencing reactions.

Principle

Several methods can be used to purify DNA from labeling reactions: precipitation, silica-resin binding, and gel filtration. Among those methods, gel filtration is the most efficient way to purify DNA from small volume of reaction mixture without losing DNA. The Ultra-Sep gel filtration material consists of spheres with uniform pores and separates molecules according molecular weight. The gel will provide excellent recovery of DNA fragments >16 base pairs or 25 mer while removing 98% of salts, dNTPs and other low molecular weight components.

Procedure

Simply apply the sequencing reaction into the Ultra-Sep™ Dye-removal Column, the dye terminators diffuse into the pores and are retained in the gel filtration material, while the labeled DNA fragments are excluded and recovered in the flow-though. The Ultra-Sep™ Dye-Removal kit is optimized for cleaning up sequencing reactions containing any type of dye terminators, such as dRhodamine and Rhodamine. This kit works well with the Tag Dyedeoxy™ and ABI Prism™ terminator cycle sequencing kits. By combining gel filtration with micro spin technology, this Ultra-Sep™ Dye-Removal kit can clean up a sequencing reaction within 7-10 minutes.

Applications

• Removal of salts and unwanted low-molecular-weight impurities
• Purification of fluorescent reaction mixtures
• Removal of free and labeled dNTPs from DNA or RNA, as in: 1. nick translation 2. end-labeling reactions 3. polymerization reaction
• Purification and desalting of proteins
• Removal of excess nucleotides and small primers in PCR reactions
• Removal of radioactive precursors
• Desalting and buffer exchange