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Sanger Sequencing Workflow
Generate high-quality Sanger sequencing data with our proven workflow
Our simple and fast Sanger sequencing workflow can be completed in less than one workday, from sample to answer. Thermo Fisher Scientific offers Applied Biosystems products to support many steps of our recommended workflow, from PCR amplification to data analysis. Learn more about each step of our recommended Sanger sequencing workflow below.
Download BigDye Terminator v3.1 protocol
Download BigDye Direct Protocol
Video: How to perform Sanger sequencing
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Workflow information
Primer Designer Tool for PCR & Sanger Sequencing
Use our online Invitrogen™ Primer Designer™ Tool to search for the right PCR/Sanger sequencing primer pair from a database of ~650,000 predesigned primer pairs for resequencing the human exome and human mitochondrial genome. Choose from different amplicon lengths to accommodate various research applications and biological sample types.
- Our primers are free of known SNPs and primer-dimers, highly target-specific and used under universal PCR conditions
- Full primer coverage for Ion Torrent™ AmpliSeq™ Exome Panel and Ion Torrent™ AmpliSeq™ Cancer Hotspot Panel v.2 Sanger confirmation workflow
- Flexible primer configuration to meet your research needs: primers can be ordered unmodified, M13-tailed, HPLC-purified or desalted
- All the primers have been checked by mass spectrometry and have passed stringent bioinformatics metrics; lab bench validation tests have shown >95% success rate
AmpliTaq Gold 360 Master Mix
AmpliTaq Gold 360 Master Mix contains the tools required for successful PCR amplification in one convenient package. All components are premixed and premeasured. AmpliTaq Gold 360 Master Mix was designed for 360° coverage of a full range of targets.
- Amplification of a broad range of targets (<3 kb) that contain high GC content, high AT content, homopolymer runs, and more
- Superior yields, allowing you to do more with your PCR amplicons
- Helps decrease false positive results with reduced bacterial background
- Reproducible results from a trusted source
ExoSAP-IT Express reagent: fastest PCR cleanup method
The Applied Biosystems ExoSAP-IT Express reagent offers rapid turnaround times and improvedefficiency of resource use while delivering the same superior cleanup as the original ExoSAP-IT reagent. Eliminate spin columns, magnetic beads, sedimentation, filtration, and gel purification. The novel ExoSAP-IT technology allows for a significant reduction in sample cleanup time with minimal steps, providing the simplest workflow.
- 5 min protocol
- One-tube, one-step PCR cleanup
- Novel enzyme technology
- 100% recovery of PCR products
Video: How to clean up PCR reactions with ExoSAP-It Express Reagent
We continue to develop superior Sanger sequencing kits that help provide solutions for all of your applications and templates.
BigDye Direct Cycle Sequencing Kit
The Applied Biosystems BigDye Direct Cycle Sequencing Kit simplifies the industry-standard Sanger sequencing workflow by combining post-PCR cleanup and cycle sequencing into a single step. The BigDye Direct Cycle Sequencing Kit requires PCR primers with M13 tails, which are available through the Primer Designer Tool.
Video: Sequencing with BigDye Direct Cycle Sequencing Kit
BigDye Terminator v3.1 Cycle Sequencing Kit
The Applied Biosystems BigDye Terminator v3.1 Cycle Sequencing Kit's robust, highly flexible chemistry is ideal for de novo sequencing, resequencing, and finishing with PCR product, plasmid, fosmid, and BAC templates.
BigDye Terminator v1.1 Cycle Sequencing Kit
The Applied Biosystems BigDye Terminator v1.1 Cycle Sequencing Kit is 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 XTerminator Purification Kit
The Applied Biosystems BigDye XTerminator Purification Kit provides a fast, simple purification method for DNA sequencing reactions to remove unincorporated BigDye terminators and salts. No more dye blobs! Cleanup is complete in under 40 min and typically requires less than 10 min of labor.
Video: How to Purify Sequencing Reactions with the BigDye XTerminator Kit
On-instrument consumables are critical components of your sequencing run. Using the right Applied Biosystems products can help reduce your re-run rate and enables high-quality, reproducible data.
Video: How to set up a Sanger sequencing or fragment analysis run
Three different polymer chemistries, each for a different need:
POP-4 Polymer
- Less viscous, fast runs
- Mostly used for human identification applications and other fragment analysis applications, and for sequencing short DNA fragments (<500 bp)
- Available for Applied Biosystems 310, 3130/xl, and 3500/xL instruments
POP-6 Polymer
- More viscous, slow runs
- Excellent resolution of nucleotides close to sequencing primer. Mostly used in conjunction with the BigDye Terminator v1.1 Cycle Sequencing Kit, for sequencing short PCR products
- Available for 310, 3130/xl, and 3500/xL instruments
POP-7 Polymer
- Mixture of PDMA and LPA
- Mostly used for sequencing of read lengths up to ~1,000 bp in conjunction with the BigDye Terminator v3.1 Cycle Sequencing Kit
- Also used for fragment analysis applications
- Available for 3130/xl, 3500/xL, and 3730/xl instruments
Minor Variant Finder Software
The improved sensitivity achieved using the FREE Applied Biosystems Minor Variant Finder Software makes Sanger sequencing the ideal choice for oncologists and/or pathologists to call low frequency somatic variants (5% or below) where the number of relevant targets is often limited. The software requires no change to your current Sanger sequencing workflow.
Benefits include:
- Call minor variants at a detection level as low as 5%
- Sequence a moderate number of targets at a low cost
- Confirm next generation sequencing findings in alignment view and Venn diagram
Video: How to detect minor variants using Sanger sequencing
Sanger Analysis Modules—free cloud-based tools
Applied Biosystems Sanger Analysis Modules are FREE innovative cloud-based secondary data analysis tools that bring together multiple data sets in one convenient place. This online solution makes it easier to view, store, and analyze Sanger sequencing data.
Key features include:
- Next-generation sequencing (NGS) confirmation—one central location to confirm your NGS variants
- Variant reporting in absolute genomic coordinates—eliminate the need to calculate from reference
- Automated database search—automatically report genomic annotations for SNPs
- .vcf output for downstream analysis—search multiple databases with the Ion Reporter annotation workflow
More about Sanger sequencing and fragment analysis software ›
Primer Designer Tool for PCR & Sanger Sequencing
Use our online Invitrogen™ Primer Designer™ Tool to search for the right PCR/Sanger sequencing primer pair from a database of ~650,000 predesigned primer pairs for resequencing the human exome and human mitochondrial genome. Choose from different amplicon lengths to accommodate various research applications and biological sample types.
- Our primers are free of known SNPs and primer-dimers, highly target-specific and used under universal PCR conditions
- Full primer coverage for Ion Torrent™ AmpliSeq™ Exome Panel and Ion Torrent™ AmpliSeq™ Cancer Hotspot Panel v.2 Sanger confirmation workflow
- Flexible primer configuration to meet your research needs: primers can be ordered unmodified, M13-tailed, HPLC-purified or desalted
- All the primers have been checked by mass spectrometry and have passed stringent bioinformatics metrics; lab bench validation tests have shown >95% success rate
AmpliTaq Gold 360 Master Mix
AmpliTaq Gold 360 Master Mix contains the tools required for successful PCR amplification in one convenient package. All components are premixed and premeasured. AmpliTaq Gold 360 Master Mix was designed for 360° coverage of a full range of targets.
- Amplification of a broad range of targets (<3 kb) that contain high GC content, high AT content, homopolymer runs, and more
- Superior yields, allowing you to do more with your PCR amplicons
- Helps decrease false positive results with reduced bacterial background
- Reproducible results from a trusted source
ExoSAP-IT Express reagent: fastest PCR cleanup method
The Applied Biosystems ExoSAP-IT Express reagent offers rapid turnaround times and improvedefficiency of resource use while delivering the same superior cleanup as the original ExoSAP-IT reagent. Eliminate spin columns, magnetic beads, sedimentation, filtration, and gel purification. The novel ExoSAP-IT technology allows for a significant reduction in sample cleanup time with minimal steps, providing the simplest workflow.
- 5 min protocol
- One-tube, one-step PCR cleanup
- Novel enzyme technology
- 100% recovery of PCR products
Video: How to clean up PCR reactions with ExoSAP-It Express Reagent
We continue to develop superior Sanger sequencing kits that help provide solutions for all of your applications and templates.
BigDye Direct Cycle Sequencing Kit
The Applied Biosystems BigDye Direct Cycle Sequencing Kit simplifies the industry-standard Sanger sequencing workflow by combining post-PCR cleanup and cycle sequencing into a single step. The BigDye Direct Cycle Sequencing Kit requires PCR primers with M13 tails, which are available through the Primer Designer Tool.
Video: Sequencing with BigDye Direct Cycle Sequencing Kit
BigDye Terminator v3.1 Cycle Sequencing Kit
The Applied Biosystems BigDye Terminator v3.1 Cycle Sequencing Kit's robust, highly flexible chemistry is ideal for de novo sequencing, resequencing, and finishing with PCR product, plasmid, fosmid, and BAC templates.
BigDye Terminator v1.1 Cycle Sequencing Kit
The Applied Biosystems BigDye Terminator v1.1 Cycle Sequencing Kit is 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 XTerminator Purification Kit
The Applied Biosystems BigDye XTerminator Purification Kit provides a fast, simple purification method for DNA sequencing reactions to remove unincorporated BigDye terminators and salts. No more dye blobs! Cleanup is complete in under 40 min and typically requires less than 10 min of labor.
Video: How to Purify Sequencing Reactions with the BigDye XTerminator Kit
On-instrument consumables are critical components of your sequencing run. Using the right Applied Biosystems products can help reduce your re-run rate and enables high-quality, reproducible data.
Video: How to set up a Sanger sequencing or fragment analysis run
Three different polymer chemistries, each for a different need:
POP-4 Polymer
- Less viscous, fast runs
- Mostly used for human identification applications and other fragment analysis applications, and for sequencing short DNA fragments (<500 bp)
- Available for Applied Biosystems 310, 3130/xl, and 3500/xL instruments
POP-6 Polymer
- More viscous, slow runs
- Excellent resolution of nucleotides close to sequencing primer. Mostly used in conjunction with the BigDye Terminator v1.1 Cycle Sequencing Kit, for sequencing short PCR products
- Available for 310, 3130/xl, and 3500/xL instruments
POP-7 Polymer
- Mixture of PDMA and LPA
- Mostly used for sequencing of read lengths up to ~1,000 bp in conjunction with the BigDye Terminator v3.1 Cycle Sequencing Kit
- Also used for fragment analysis applications
- Available for 3130/xl, 3500/xL, and 3730/xl instruments
Minor Variant Finder Software
The improved sensitivity achieved using the FREE Applied Biosystems Minor Variant Finder Software makes Sanger sequencing the ideal choice for oncologists and/or pathologists to call low frequency somatic variants (5% or below) where the number of relevant targets is often limited. The software requires no change to your current Sanger sequencing workflow.
Benefits include:
- Call minor variants at a detection level as low as 5%
- Sequence a moderate number of targets at a low cost
- Confirm next generation sequencing findings in alignment view and Venn diagram
Video: How to detect minor variants using Sanger sequencing
Sanger Analysis Modules—free cloud-based tools
Applied Biosystems Sanger Analysis Modules are FREE innovative cloud-based secondary data analysis tools that bring together multiple data sets in one convenient place. This online solution makes it easier to view, store, and analyze Sanger sequencing data.
Key features include:
- Next-generation sequencing (NGS) confirmation—one central location to confirm your NGS variants
- Variant reporting in absolute genomic coordinates—eliminate the need to calculate from reference
- Automated database search—automatically report genomic annotations for SNPs
- .vcf output for downstream analysis—search multiple databases with the Ion Reporter annotation workflow
More about Sanger sequencing and fragment analysis software ›
Sanger sequencing chemistry guide
A perfect handbook for scientists and researchers using Sanger sequencing. In this chemistry guide, you will learn the basic principles of Sanger sequencing from workflow to applications.
- Sanger sequencing applications
- DNA template preparation
- Cycle sequencing
- Purification of extension products
- Capillary electrophoresis
- Data analysis
- Troubleshooting
For Research Use Only. Not for use in diagnostic procedures.