High quality sequence data obtained using the mitoSEQr™ resequencing system (Figure 1).
Experimental Workflow: Find the Products You Need for Every Step
PCR Amplification & Cleanup
Sequencing Reaction Cleanup
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Step 1: DNA Purification
DNA extraction is a critical first step in the experimental workflow of DNA Sequencing and Fragment analysis. The overall quality, accuracy and length of the DNA sequence read can be significantly affected by characteristics of the sample itself, and the method chosen for nucleic acid extraction. Ideal methods will vary depending on the source or tissue type, how it was obtained from its source, and how the sample was handled or stored prior to extraction.
Products for Step 1: DNA Purification
Step 2: PCR Amplification & Cleanup
Many common DNA sequencing workflows require you to amplify your extracted DNA sample before sequencing. To amplify your DNA sample, you need DNA polymerase (such as AmpliTaq® Gold DNA Polymerase), nucleotides (dNTPs), reaction buffer, primers, and a thermal cycler. Sequences can be obtained from NCBI Probe web site.
Products for Step 2: PCR Amplification & Cleanup
- Custom Oligo Synthesis - Unlabeled Oligo
We offer a portfolio of fluorescent dyes for gene expression studies, 5’ nuclease assays, PCR, fragment analysis, and genotyping. When you know the exact sequence of a fluorescent labeled probe or primer, we can synthesize it for you.
Step 3: Cycle Sequencing
Cycle sequencing is a simple method in which successive rounds of denaturation, annealing, and extension in a thermal cycler result in linear amplification of extension products. The products are then injected into a capillary. All current Applied Biosystems DNA sequencing kits use cycle sequencing protocols. For de novo sequencing we recommend Dye Terminator chemistry.
Products for Step 3: Cycle Sequencing
- BigDye® Terminator v1.1 Cycle Sequencing Kits
BigDye® Terminator v1.1 Cycle Sequencing Kits are designed for specialty applications that require optimal basecalling adjacent to the primer and for sequencing short PCR product templates with rapid electrophoresis run modules.
- BigDye® Terminator v3.1 Cycle Sequencing Kits
The BigDye Terminator v3.1 Cycle Sequencing Kit's robust, highly flexible chemistry is ideal for de novo, resequencing, and finishing with PCR product, Plasmid, Fosmid, and BAC templates.
- dRhodamine Terminator Cycle Sequencing Kits
The dRhodamine Terminator Cycle Sequencing Kit provides optimized dRhodamine dideoxy terminators chemistries for decreased background noise in a wide array sequencing applications.
- dGTP BigDye® Terminator Cycle Sequencing Kits
The dGTP BigDye® Terminator Cycle Sequencing Kit's fluorescent dideoxy terminator chemistry is optimized for sequencing through GT- and G-rich templates.
Step 4: Sequencing Reaction Cleanup
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.
Products for Step 4: Sequencing Reaction Cleanup
- Centri-Sep™ Columns
Centri-Sep™ spin columns are used for the fast and efficient purification of nucleic acids. The Centri-Sep gel provides excellent recovery of DNA fragments larger than 16 base pairs, while removing greater than 98% of salts, NTP's and other unwanted low-molecular-weight impurities
- Centri-Sep™ 96-Well Plates
Centri-Sep™ 96's hydrated matrix enables effective and reliable high-throughput removal of excess BigDye® terminators from DNA sequencing reactions, giving you clean sequencing reads with uniform signal intensity starting with the first base.
- Centri-Sep™ 8-Well Strips
Centri-Sep™ 8's convenient pre-hydrated 8-well strips enable you to remove excess BigDye® terminators from sequencing reactions, unwanted salts, low-molecular-weight impurities, free and labeled dNTPs, and excess nucleotides and small primers from PCR reactions. You can also used the strips to purify proteins.
- BigDye XTerminator® Purification Kit
The BigDye XTerminator® Purification Kit is a fast, simple purification method for DNA sequencing reactions that removes unincorporated BigDye® terminators.
Step 5: Capillary Electrophoresis
After performing the post-sequencing reaction purification, samples are ready for analysis on an Applied Biosystems capillary electrophoresis-based genetic analyzer. During capillary electrophoresis, the products of the cycle sequencing reaction migrate through capillaries filled with polymer. The negatively charged DNA fragments are size separated as they move through the polymer in the capillaries toward the positive electrode.
Products for Step 5: Capillary Electrophoresis
- 3500 Genetic Analyzer
The 3500 8-capillary platform can run a wide variety of applications—including de novo sequencing and resequencing (mutational profiling)—as well as microsatellite analysis, MLPA™, LOH, MLST, AFLP® and SNP validation or screening. The majority of applications can be run on a single polymer and capillary array.
- 3500xl Genetic Analyzer
The 3500xL 24-capillary platform can run a wide variety of applications, including de novo sequencing and resequencing (mutational profiling), as well as microsatellite analysis, MLPA™, LOH, MLST, AFLP® and SNP validation or screening. The majority of applications can be run on a single polymer and capillary array.
- 3730 DNA Analyzer
The 48-capillary 3730 DNA Analyzer is the Gold Standard in medium-to-high throughput genetic analysis. Use this for DNA fragment analysis applications such as microsatellites, AFLP, SNP analysis, mutation detection and traditional DNA sequencing. Get the highest quality data at a low cost per sample. This is upgradeable to 96 capillaries.
- 3730xl DNA Analyzer
The 96-capillary 3730xl DNA Analyzer is the Gold Standard for high throughput genetic analysis. Use this for DNA fragment analysis applications such as microsatellites, AFLP, SNP analysis, mutation detection and traditional DNA sequencing. Get the highest quality data at a low cost per sample.
- 3130 Genetic Analyzer
Applied Biosystems 3130 Genetic Analyzer is the latest generation of 4-capillary electrophoresis instruments for the low to medium throughput laboratories. The system offers industry-leading performance, plus sophisticated automation capabilities allowing you to save time, reduce costs and increase productivity.
- 3130xl Genetic Analyzer
Applied Biosystems 3130xl Genetic Analyzer is the latest generation of 16-capillary electrophoresis instruments for the medium throughput laboratories.
- 310 Genetic Analyzer
The ABI PRISM® 310 Genetic Analyzer is an automated single-capillary genetic analyzer designed for a wide range of sequencing and fragment analysis applications.
Step 6: Data Analysis
After electrophoresis, Data Collection software creates a Sample File of the raw data. Using downstream software applications, further data analysis is required.
Products for Step 6: Data Analysis
- SeqScape® Software
Applied Biosystems SeqScape® software is designed for reference based analysis such as mutation detection and analysis, SNP discovery and validation, pathogen sub-typing, allele identification, and sequence confirmation. This product is also integrated for use with VariantSEQr™ Resequencing Set.
- Variant Reporter® Software
The Variant Reporter™ Software performs comparative sequencing, also known as direct sequencing, medical sequencing, PCR sequencing and resequencing with DNA sequencing files. The software is designed for reference based and non-reference based analysis such as mutation detection and analysis, SNP discovery and validation and sequence confirmation.