Millipore Technical Publications |
 | PO- GSAC 2001 | |
Integrating Sequencing Reaction Purification with the ABI Prism 3700 DNA Sequencer | ||
| Lit No: | GSAC 2001 | |
| Year: | 2001 | |
| The ABI PRISM 3700 capillary sequencer, combined with the new BigDye Terminator v3.0 Cycle Sequencing Kit (Applied Biosystems) provides the most high throughput, automated and robust solution for production DNA sequencing available. As a consequence, labor-intensive sample preparation methods upstream of sequencing can quickly become the most costly and rate-limiting steps as genomics facilities scale-up. One time-tested and reliable way to increase productivity is to integrate sample processing steps with analysis. The new Millipore Direct-Inject holder was designed to fit on the deck of the ABI 3700, to eliminate processing steps, and to fully realize the quality and throughput enhancements associated with the new sequencing chemistry. Montage SEQ384 plates (Millipore) were used to purify 1/8th scale BigDye v3.0 sequencing reactions on a Beckman Multimek or Biomek FX liquid handler. After filtration, a small volume of injection solution was dispensed to each well of the Montage SEQ384 plate, the plate was sealed with tape, and transferred to the Direct-Inject holder. The Direct-Inject holder was placed at one position on the ABI 3700 deck, and the purified sequencing products were automatically transferred from the Montage SEQ384 plate by the ABI 3700 onto the loading bar, and electrokinetically injected into the capillary array. Four Montage SEQ384 plates containing purified sequencing reactions can be loaded onto “Direct-Inject holders on the ABI 3700, and sixteen consecutive runs can be carried out without user intervention. Details of the new sample preparation process, and an analysis of the quality of the DNA sequencing results will be shown. | |
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