Sequencing Reaction Clean Up
The chemagic SEQ Pure Kit special was developed to purify products from sequencing reactions, including BigDye® terminator reactions. Purification is performed using chemagen´s unique Magnetic Beads and optimised binding and wash buffers, The purification process is simple, fast and convenient as it requires no centrifugation or filtration steps and removes any type of dye terminator. Kit versions are available for being used on the chemagic Magnetic Separation Module I or any liquid handling platform.
The kit has been optimised for use with Amershams DYEnamic™ ET Dye Terminator Kit and ABI BigDye® chemistries (including v1.1 and 3.1) and yields highly purified sequencing reaction products. The protocol recommends 1/4 - 1/8 x scale reactions to save on reagent costs, reduce dye blobs and provide for more usable sequencing data. There is no need to adjust the protocol for variations in dye terminator or sample volume. The obtained results are of the highest quality with high fluorescent signal intensities, routinely long read lengths (> 900 bp), 98 % accuracy to 900 bases and Phred20 quality scores = 825 bases. In combination with the chemagic Magnetic Separation Module I an automated system validated for high throughput is available. The chemagic SEQ Pure Kit special can also be used with commercially available liquid handling systems.
The following chromatogram shows a purified ABI BigDye terminator v3.1 sequencing reaction using the chemagic SEQ Pure Kit special and the chemagic MSM I.
Sequencing reactions were performed using 1/4 x BigDye terminator v3.1 chemistry, pGEM-3Zf(+) (400 ng), and pUC/M13 (–21) oligonucleotide primer. After purification, the samples were analyzed on an Applied Biosystems 3730 DNA Analyzer. This electropherogram is typical of the high quality reads achieved using this cleanup method (Phred20 = 825).
