Sanger Sequencing Reaction Purification

After the sequencing reaction, it is important to remove unincorporated dye terminators and salts that may compete for capillary electrophoretic injection. Unincorporated terminators can co-migrate with the sequencing template, resulting in basecalling errors, and excess salt translates to poor signal-to-noise ratios.

Whereas ethanol precipitation methods can be sensitive to variations in template quality, the BigDye® XTerminator™ Purification Kit provides a robust chemistry, that will yield consistent, high-quality template agnostic of template type or concentration. The BigDye XTerminator bead-based reagent sequesters sequencing reaction impurities including salt ions, unincorporated dyes, and dNTPs to prevent co-injection with dye-labeled extension products during capillary electrophoresis.

We recommend the use of the BigDye® XTerminator™ Purification Kit for sequencing template cleanup. BigDye XTerminator is a fast, simple purification method for sequencing reactions that affords high-quality data with long reads, strong signal, and superior short fragment recovery. Cleanup is complete in 40 minutes with less than 10 minutes of hands-on labor and requires no liquid transfer (Figure 1). The resulting purified samples will be free of dye blobs, and are stabilized, which is helpful if capillary electrophoresis will not immediately follow cleanup. The easy-to-follow procedure is amenable to 96-well and 384-well format procedures. Sealed reaction plates are stable for 48 hours at room temperature or 10 days at 4 °C.