Sanger Sequencing Workflow | Thermo Fisher Scientific

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.

Video: How to Perform Sanger Sequencing

Workflow

PCR amplification

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

Visit tool ›

Platinum II Taq Hot-Start DNA Polymerase

Invitrogen Platinum II Taq Hot-Start DNA Polymerase helps you get to sequencing reactions faster. A unique combination of innovative buffer, high-performance Taq DNA polymerase, and superior Platinum hot-start technology helps enable exceptional PCR results, even in the toughest applications.

Universal primer annealing at 60°C enables co-cycling of all assays, reducing tedious optimization steps
An engineered Taq polymerase enables 4X faster DNA synthesis, inhibitor resistance, and robust amplifi cation
Platinum hot-start technology offers superior specificity, sensitivity, and yields, and allows for room temperature reaction setup
2X master mix format helps reduce pipetting errors with fewer pipetting steps

More formats and information are available at thermofisher.com/platinumiitaq.

PCR cleanup

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

Learn more about ExoSAP-IT Reagents ›

Also available:
Original ExoSAP-IT reagent
HT ExoSAP-IT High-Throughput PCR Product Cleanup

Cycle sequencing

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

Created by experts and refined by global teams, the BigDye Terminator v3.1 cycle sequencing kit offers exceptional versatility and high performance. This is a comprehensive, exhaustive solution, built for de novo sequencing, resequencing, and finishing with PCR product, plasmid, fosmid, and BAC templates.

Improved quality of results and success rates, particularly with challenging templates

Suitable for a wide range of sequencing applications
Optimized for long read lengths
Sophisticated dye mobility characteristics
Refined performance reading through GT-rich regions
Optimal signal balance
Rapid completion of sequencing projects

Learn more about BigDye Terminator v3.1 Cycle Sequencing Kit ›

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 basescalling adajcent to the primer, and for sequencing short PCR product templates with rapid electrophoresis run modules.

Learn more about BigDye Terminator v1.1 Cycle Sequencing Kit ›

Sequencing cleanup

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

Learn more about BigDye XTerminator Kit ›

Capillary electrophoresis

A range of platforms to meet your throughput needs

Throughput	Low	Medium	Medium	High
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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.

SeqStudio Cartridge

The Applied Biosystems SeqStudio Cartridge is an easy-to-use reagent cartridge that includes capillaries, POP-1 universal polymer, buffer, and pump. The POP-1 universal polymer allows for flexibility to perform Sanger sequencing and fragment analysis on the SeqStudio Genetic Analyzer System with one cartridge. Just load your samples, click in the cartridge, and go.

Browse other on-instrument consumables

Capillaries
Polymers
Buffers & chemicals

Video: How to set up a Sanger sequencing or fragment analysis run

Data analysis

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 Kit

The Applied Biosystems Sanger Sequencing Kit provides a convenient and affordable solution for preparing sequencing reactions. The kit provides all reagents needed for PCR cleanup, cycle sequencing, and sequencing product cleanup. Learn more ›

What makes a gold standard sequencing product?

Gold standard represents quality and dependability. We commit to quality to help ensure results you can rely on.
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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.