DNA Purification Step with Sanger Sequencing
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.
BigDye® XTerminator™ Purification
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 48hours at room temperature or 10 days at 4 °C.
We also offer the Centri-Sep™ system for sample cleanup in several configurations: individual columns, 8-well strips, and 96-well plates (see table below). The column design is based on the description of gel filtration for purification of DNA from nick translation reactions by Sambrook et. al. Each Centri-Sep unit consists of a specially fitted microfuge tube, dry gel, wash tube, and sample collection tube.
Product Selection Guide: Sample Cleanup
|Cleanup Method||Description & Application||Product|
|Reagent-Based Sequencing Template Cleanup||Scavenges all unincorporated BigDye® terminators. Stabilizes samples before analysis. Best for long and short fragment recovery.|
|Gel Filtration Spin Columns||Recovers DNA fragments larger than 16 base pairs. Removes > 98% of salts, NTP's and other unwanted low-molecular-weight impurities.|