Frequently Asked Questions
Template Preparation
What templates can be used for Sanger sequencing?
Multiple types of templates can be used for Sanger sequencing. Both double-stranded and single stranded DNA can be sequenced. The most commonly used DNA templates include: plasmid, PCR products, and rolling cycle amplification products. In addition, bacterial artificial chromosome, genomic DNA, and cosmid DNA have also served as templates.
Why do I need to purify PCR product template?
It is necessary to purify PCR product templates because after a PCR reaction, unincorporated dNTPS, primers and DNA polymerases remain in the reaction. Without purification or cleanup, these contaminants may change or interfere with the cycle sequencing reactions,resulting in suboptimal or undesired extension products.
How do I purify the PCR product template?
There are different methods for purification of PCR products, including enzyme treatment, binding to spin columns or magnetic beads or ethanol precipitation. Our ADS™ PCR cleanup beads are cost-effective for PCR product purification.
Cycle Sequencing
What are SupreDye™ Cycle Sequencing Kits?
SupreDye™ Cycle Sequencing Kits are our proprietary Sanger sequencing kits that are powerful alternatives to BigDye® Terminator Cycle Sequencing kits with similar performance. The SupreDye™ Cycle Sequencing Kits can be successfully used on ABI 310, 3130 series, and 3730 series of Genetic Analyzers without alteration to the BigDye’s experimental protocols or other reagents.
What types of SupreDye™ Cycle Sequencing Kits are available?
Four types of SupreDye™ Cycle Sequencing Kits are available. The table below lists these kits and their uses.
| SupreDye™ Cycle Sequencing Kits | BigDye Alternatives | Applications |
| v1.1 | BigDye Terminator v1.1 | Optimized for short read or near primer reading; for regular template |
| v3.1 | BigDye Terminator v3.1 | Long read for de novo sequencing or resequencing, regular andlarger template |
| dGTP v1.1 | dGTP BigDye Terminator v.1.0 | Optimized for short read or near primer reading; for high G content (GC or GT) templates |
| dGTP v3.1 | dGTP BigDye Terminator v.3.0 | Long read for de novo sequencing or resequencing, high G content (GC or GT) templates |
How should I use SupreDye™ Cycle Sequencing Kits?
Use the SupreDye™ Cycle Sequencing Kits and 5X Sequencing Reaction Buffer as an alternative to BigDye Terminator Cycle Sequencing Kits and 5X Sequencing Buffer. All other reagents and protocols remain the same. SupreDye™ Cycle Sequencing Kits can also be used with other ADS™ reagents.
How do I store the SupreDye™ Cycle Sequencing Kits?
The SupreDye™ Kits are shipped with dry ice and should be stored at -20°C after arrival.
Sequencing Reaction Cleanup
Why do I need to clean up sequencing reactions?
Completed sequencing reactions contain unincorporated dNTPs, ddNTPs, primers, and enzyme components. If these components, particularly the fluorescently labeled ddNTPs, are loaded into the capillary electrophoresis, they can interfere with the migration of extension products and signal detection or basecalling, which can result in issues like dye blobs.
How do I clean up sequencing reactions?
There are several methods for purifying sequencing reactions, including size exclusion chromatography, affinity purification, magnetic bead cleanup, and ethanol precipitation. Any of these methods can be chosen for the purification process. We offer the SupreDye™ XT Purification Kit as an alternative to the XTerminator Purification Kit, as well as the ADS™ Sequencing Reaction Cleaning Beads, for the effective removal of contaminants from sequencing reactions.
How should I choose between the SupreDye™ XT Purification Kit and ADS™ Sequencing Reaction Cleaning Beads?
While both products effectively remove contaminants to achieve optimal sequencing results, they operate through different mechanisms and produce distinct end results. The SupreDye™ XT Purification Kit binds to the contaminants, whereas the magnetic beads bind to the extension products. The SupreDye™ XT Purification Kit protocol does not include a wash step, leading to better recovery of extension products; samples are loaded directly onto the sequencer after the mixing step. The ADS™ Sequencing Reaction Cleaning Beads are optimized for dye-blob removal with a minimal wash step, yielding good recovery of extension products. For those with budget constraints, magnetic beads are a more cost-effective option.
Sample Loading and Capillary Electrophoresis
How are samples loaded onto capillary electrophoresis instruments?
The method used for sample loading depends on the sequencing reaction purification technique. If the SupreDye™ XT Purification Kit is used, the sample is loaded directly onto the sequencer. If products are purified using ethanol, they are typically resuspended in a highly deionized formamide, such as TruPure™ Formamide. Resuspending samples in water can cause C peak tailing, a problem that can be resolved by using formamide instead.
How is TruPure™ Formamide different from other formamides?
TruPure™ Formamide is a highly deionized and purified solution that increases the intensity and stability of sequencing signals. It has been successfully used as a direct alternative to Hi-Di™ formamide.
Why are different polymers used as separation matrices for sequencing?
The choice of polymer depends on specific sequencing requirements, such as achieving long or short reads, and compatibility with the sequencing instrument. Our PwrPOP™-7 Sequencing Polymer is the only polymer that can be used on the 3730 series to achieve optimal long reads.
How are PwrPOP™-4, -6, or -7 polymers used?
PwrPOP™-4, -6, and -7 polymers are high-performance replacements for POP-4™, POP-6™, and POP-7™, respectively. They can be used with the same protocols without requiring any operational changes. These polymers ensure high-quality separation of extension products, resulting in evenly spaced peaks and long read lengths.
Capillary Array Regeneration
Why should I regenerate my capillary array?
Over hundreds of runs, polymer debris and proteins can accumulate on the capillary array. This buildup can degrade the separation performance of the polymers, resulting in issues with peak spacing and read length. Regenerating the capillary array is a necessary maintenance step to restore its optimal performance without the need for a full replacement.
What should I use to regenerate a capillary array?
The ADS™ Capillary Array Regeneration Kit is designed to restore the performance of capillary arrays for all ABI genetic analyzers. Simply follow the detailed protocol included with the product to achieve optimal results.
When should I regenerate my capillary array?
A capillary array performing optimally will produce uniform read lengths and peak spacing (though specific values may vary between arrays). After several hundred uses, the accumulation of contaminants can lead to shorter read lengths, wider peak spacing, and tailing peaks in all four colors, even with regular cleaning. These are clear indicators that the array needs regeneration. We recommend regenerating the capillary array with our Capillary Array Regeneration Kit every 500 runs.
How does the cost of regeneration compare to replacement?
Regenerating a capillary array is significantly more cost-effective than replacing it. The cost of regeneration is only a fraction of the cost of a new array.
How do I regenerate a capillary array?
The ADS™ Capillary Array Regeneration Kit includes a detailed protocol that provides step-by-step instructions for cleaning and regenerating the array.
How long will a capillary array last after regeneration?
A regenerated array can last for hundreds of additional runs, with performance life depending on instrument maintenance. Our data indicates that a regenerated array can typically be used for another 500 runs before needing subsequent regeneration. An array can be regenerated multiple times before a replacement is necessary.
