BigDye™ Terminator v3.1 Cycle Sequencing Kit, 100 Reactions - FAQs

When contacting Genetic Analysis Support (FAS, FSE, Technical Support), please have (if possible):

Instrument model and serial numbers
Version of the software are you using
Application and/or kit information
Kit lot number
Number of runs on the capillary or array
Polymer type and lot number
Buffer lot number
Hi-Di Formamide lot number or loading solution information
Sample data that can be sent to the support person
Details on:
  -Reaction setup (i.e., how much DNA was used, primer, etc.)
  -How the reactions were cleaned up (alcohol precipitation, columns, etc)
  -What template DNA was used (i.e. plasmid, PCR product)
  -How the template DNA was prepared?

All of this information will help the support person quickly and accurately assess the problem and provide you with recommendations for resolution.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

In order to get through homopolymeric regions, you can either use anchored sequencing primers or try using the dRhodamine Terminator Cycle Sequencing Kit since it uses ddTTP instead of ddUTP and has been shown to be less prone to producing stutters, specifically with poly-T regions.

For more information on sequencing through homopolymer stretches, please refer to the DNA Sequencing by Capillary Electrophoresis: Applied Biosystems Chemistry Guide: Second Edition (Cat. No. 4305080, Rev. C). The guide can be found by searching the Thermo Fisher Scientific website with the catalog number 4305080.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

When trying to sequence through GC-rich regions, the following tips may improve your success:

• Set the reaction up at 1X or 0.5X strength. Heavily diluting the BigDye Terminator Ready Reaction mix can reduce the success of the sequecing reaction through long GC stretches.

• Perform a hot start at 98–99°C for 5 minutes.

• Use 5% (w/v) DMSO or freshly made betaine in the reaction.

• Use the dGTP BigDye Terminator Cycle Sequencing Kit. G peaks will appear compressed due to the presence of ddGTP, but sequencing through long GC stretches using the dGTP kit is typically more successful than with the standard BigDye Terminator Cycle Sequencing Kits (containing ddITP). For more information on sequencing GC-rich DNA, please refer to the DNA Sequencing by Capillary Electrophoresis: Applied Biosystems Chemistry Guide: Second Edition (Cat. No. 4305080, Rev. C). The guide can be found by searching the Thermo Fisher Scientific website with the catalog number 4305080.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Some of the causes of another sequence appearing under the primary sequence are:

• Contamination: There is more than one species of DNA present (e.g., multiple PCR products).

• Primers: Primer is annealing in more than one location on the template, primer dimer, primer degradation or not manufactured properly resulting in N+1 or N-1, carry over from PCR reaction, or primers pairs in a multiplex reaction that are not appropriate for multiplexing (i.e., primers anneal inappropriately).

• Spectral/Matrix: If the raw data signal intensity of the sample is too high or saturated it can exceed the amount of color bleedthrough (or spectral overlap) that the matrix (310) or spectral (3130, 3730, 3500) are removing, resulting in secondary peaks appearing in a very specific pattern (e.g., a red peak always appearing under a green peak). A change in the camera, laser, or optical alignment requires that a new matrix be made or a new spectral calibration be performed.

For more information on more than one sequence or set of peaks in a sequencing run, please refer to the DNA Sequencing by Capillary Electrophoresis: Applied Biosystems Chemistry Guide: Second Edition (Cat. No. 4305080, Rev. C). The guide can be found by searching the Thermo Fisher Scientific website with the catalog number 4305080.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

The cause of this is usually an overabundance of template DNA relative to the amount of BigDye Terminator Ready Reaction mix being used in the reaction. This can drive the reaction to incorporate the labeled ddNTPs closer to the 5’ end of the sequence. To balance the signal, either increase the amount of BigDye Terminator Ready Reaction mix being used in the reaction or decrease the concentration of the template DNA.

For more information on other causes of short reads and how to address each issue, please refer to the DNA Sequencing by Capillary Electrophoresis: Applied Biosystems Chemistry Guide: Second Edition (Cat. No. 4305080, Rev. C). The guide can be found by searching the Thermo Fisher Scientific website with the catalog number 4305080.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Some of the causes of dye breakdown or artifacts in the data are:

• Oxidation or change in pH: Injecting out of water or old Hi-Di Formamide that has broken down. Also, some cleanup methods can oxidize the dyes.

• Array: An old or contaminated array can cause dye breakdown.

• Arcing: An arcing event on the system can destabilize the blue baseline and—in severe cases—mask it completely.

For more information on other causes of dye breakdown and artifacts in data and how to address each issue, please refer to the DNA Sequencing by Capillary Electrophoresis: Applied Biosystems Chemistry Guide: Second Edition (Cat. No. 4305080, Rev. C). The guide can be found by searching the Thermo Fisher Scientific website with the catalog number 4305080.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

A change in the read length is usually caused by something that either changes the migration of the DNA or the efficiency of the sequencing reaction. Some of the things that can cause this are:

• Buffer: Buffer may be prepared incorrectly or on the system for too long.

• Polymer: Polymer may have been on the system the system too long, used past the expiration date, frozen/crystallized and thawed, or mixed with water or buffer during capillary fill. There may be a mismatch between polymer type selected in software and what is installed on the system.

• Environment: Instrument may be operating outside environmental specifications in the Site Preparation Guide. You may have poor/improper instrument ventilation, or air flow may be blowing directly on the system.

• DNA quality: RNA/protein contamination may be reducing the sequencing reaction efficiency. If working with PCR product, there may be carryover of primers and/or dNTPs from the PCR reaction, or the sequencing reaction cleanup is incomplete. If using a kit that has beads in it for the purification, beads may be blocking the capillary tip. If using BigDye XTerminator Purification, heating the XTerminator reagent can cause loss of smaller products.

• DNA quantity: There may be too much DNA competing for entry into the capillary with labeled product, or excessive amounts of DNA creating a temporary blockage of the capillary.

For more information on other causes of short reads and how to address each issue, please refer to the DNA Sequencing by Capillary Electrophoresis: Applied Biosystems Chemistry Guide: Second Edition (Cat. No. 4305080, Rev. C). The guide can be found by searching the Thermo Fisher Scientific website with the catalog number 4305080.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Capillary electrophoresis systems are complex systems, and troubleshooting data quality can be complicated. The biggest contributors to data quality can be divided into these areas: software, chemistry, and instrument performance.

Troubleshooting software
Ensure that the run module you are using and the dye set match the chemistry and instrument setup. For example, if you are running a multicapillary instrument with a 50 cm array on it, the run module should have a “50” in the name. On a multicapillary system, BigDye Terminator v3.1 should run under Dye Set Z, BigDye Terminator v1.1 chemistry should run in Dye Set E (on the ABI PRISM 310 Genetic Analyzer, both chemistries run under Filter Set E, but require different matrices for analysis). The mobility file (DyeSet/Primer) should have the correct instrument, polymer, and chemistry in the name (e.g. KB_3130_POP7_BDTv.3.mob).

Troubleshooting chemistry and instrument performance
•Sequencing Standards: validate instrument
If Sequencing Standards fail, it suggests a possible electrophoresis problem due to array, polymer, buffer, water, septa, plastics, or maybe a hardware failure such as autosampler, laser, camera, etc.

•pREF-BDT Control DNA: validate cycle sequencing and its clean-up
If the pREF-BDT control reactions fail and the Sequencing Standards pass, look into potential problems with the sequencing kit, thermal cycler, and cleanup procedure.
The pREF-BDT Control DNA and M13 (–21) primers needed to run the reactions are included with each BigDye Terminator Cycle Sequencing Kit

•Custom Internal Controls: validate PCR and its clean-up
If the Sequencing Standards and the pGEM controls passed, then look into potential problems with template quantity and quality, primers, PCR reaction and purification.

For more information on troubleshooting your data, please refer to the DNA Sequencing by Capillary Electrophoresis: Applied Biosystems Chemistry Guide: 3rd Edition, which can be downloaded from this link (https://www.thermofisher.com/us/en/home/global/forms/sanger-sequencing-guide-download-form.html).

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

The BigDye Terminator v1.1 and v3.1 chemistries can be diluted, and a 5X Sequencing Buffer is provided with the kit to enable you to do so. However, dilution of the BigDye Terminator may compromise your data integrity depending on the cycle sequencing protocol you are following and the sequence of the particular DNA you are investigating. TheBigDye Direct Cycle Sequencing Kit and the dGTP BigDye Terminator Ready Reaction Kits v1.0 and v3.0 cannot be diluted.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

After the cycle sequencing reaction is complete, unincorporated ddNTPs can form complexes that migrate more slowly than individual ddNTPs would. These complexes can interfere with electrokinetic injection, electrophoretic separation, and data analysis. In the electropherogram these can be observed as a region containing multicolored peaks of varying sizes or blobs, depending on the efficiency of the cleanup. In BigDyeTerminator v3.1 reactions, these blobs may appear around 70–80 bp and, in severe cases, 110–120 bp. For BigDye Terminator v1.1, the blobs will either migrate ahead of the data collection time (so they will not be visible) or they may be visible at the very beginning of the run.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

While there are many kits and methods available to clean up the extension products from a sequencing reaction, the ones that Thermo Fisher Scientific has formally tested and recommends are:

•BigDye XTerminator Purification Kit
•Ethanol precipitation
•Spin column/plate purification (e.g. Centri-Sep columns, DTR Kits from Edge Biosystems)

A list of alternative methods for purification is available in the DNA Sequencing by Capillary Electrophoresis: Applied Biosystems Chemistry Guide: Second Edition (Cat. No. 4305080, Rev. C, page 119). The guide can be found by searching the Thermo Fisher Scientific website with the Cat. No. 4305080.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

BigDye Direct Cycle Sequencing Kit supplies reagents for the complete cycle sequencing workflow, including the PCR reaction, reaction cleanup, and sequencing the PCR product. The post-PCR and cycle sequencing process is combined into a single step, and the workflow requires that the initial PCR primers be designed with an M13 tail, since M13 primers will be used for the sequencing reaction. BigDye Terminator Cycle Sequencing Kits are designed so that the initial PCR and the cleanup are performed as separate reactions. This allows the user more flexibility in primer design and the option to break up the workflow. For the comparative studies that we performed in-house, we found sequencing data obtained using these two kits to be concordant and of at least equivalent quality (typically the BigDye Direct Cycle Sequencing Kit produced better results).

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

The formulation of the 5X Sequencing Buffer that comes with the BigDye Terminator Kit is proprietary.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

The BigDye Terminator Ready Reaction Mix contains the dNTPs, fluorescently labeled ddNTPs, buffer, and other components that are needed to drive the sequencing reaction. The specific composition of the BigDye Terminator Ready Reaction Mix is proprietary.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Selecting the best kit for your application depends on what you are trying to do—for example, the goals and needs for a resequencing project may be different than for SNP detection. To help guide you, Thermo Fisher Scientific has put together an Applications/Chemistry table found in the DNA Sequencing by Capillary Electrophoresis: Applied Biosystems Chemistry Guide: Second Edition (Cat. No. 4305080, Rev. C). The guide can be found by searching the Thermo Fisher Scientific website with the Cat.No 4305080.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

The BigDye Terminator v1.1 kit is optimized to produce better incorporation close to the primer and is typically able to produce sequence out to approximately 700–750 bp under optimal conditions (e.g., a well-maintained instrument, clean DNA). The BigDye Terminator v3.1 kit is optimized for longer reads, but the resolution in the first 30–50 bp may be of low quality. Under optimal conditions (e.g., a well-maintained instrument and clean DNA), The BigDye Terminator v3.1 kit is typically able to produce sequence out to approximately 1,200 bp.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

The ddNTPs in the two kits are labeled with different dyes that, while spectrally similar, do require a different Dye Set on our multicapillary systems and a different matrix on our single-capillary system. The ratio of dNTP/ddNTPs is also different to allow better incorporation of ddNTPs closer to the primer in the case of the BigDye Terminator v1.1 kit or a longer read in the case of BigDye Terminator v3.1 kit.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

The concentration of the pREF-BDT control is 200 ng/µL. The concentration of the M13 primer is 0.8 pmol/µL.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

We recommend the BigDye Terminator v3.1 Cycle Sequencing Kit if you would like to use custom PCR primers instead of M13 primers for sequencing.

The BigDye Direct kit must use the M13 primers provided in the kit for sequencing, as the kit contains the PCR and Sequencing components and are optimized to work together.

The BigDye Terminator v1.1 & v3.1 5X Sequencing Buffer is not available as a standalone item. It is only available as part of the BigDye Terminator v3.1 Cycle Sequencing Kit.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

We do not sell the -21 M13 forward control primer separately, however we provide the primer sequence for your convenience: TGTAAAACGACGGCCAGT

Please feel free to use our oligo services to order this primer (https://www.thermofisher.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos.html).
We also offer an -21M13 forward primer that is one basepair shorter than that contained in the kit (Cat. No. N52002).

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

We do not sell the pREF-BDT control separately from the DNA sequencing kit.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

The pGEM control is 200 ng/µL and the M13 primer 0.8 pmol/µL

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

The control is pREF-BDT control plasmid DNA which serves as a double-stranded control

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

The calculation can be done manually or by using our Applied Biosystems BigDye Terminator v1.1 & v3.1 5X Sequencing Buffer Calculator.

The formula is provided below:

Volume of BigDye Terminator v1.1 & v3.1 5X Buffer (µL) = 0.5 x [(total reaction volume/2.5) - volume of BigDye Terminator Ready Reaction Mix].

For example, if you are using 2 µL of the BigDye Terminator Ready Reaction Mix and a 15 µL reaction volume

Volume of BigDye Terminator v1.1 & v3.1 5X Buffer (µL) = 0.5 x [(15/2.5) - 2] = 2 µL

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

We provide the BigDye Terminator v1.1 & v3.1 5X Buffer to enable dilution of the BigDye Terminator Ready Reaction Mix. However, without optimization, diluting the BigDye Terminator Ready Reaction Mix may cause deterioration of sequencing quality. We cannot guarantee the performance of BigDye chemistry when it is diluted.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

The BigDye Terminator v1.1 & v3.1 5X Buffer is used to maintain the optimal buffer concentration in the sequencing reaction when less than the recommended volume of BigDye Terminator Ready Reaction Mix is used. The BigDye Terminator v1.1 & v3.1 5X Buffer amount varies depending on the volume of the BigDyeTerminator Ready Reaction Mix. To determine the amount of BigDye Terminator v1.1 & v3.1 5X Buffer, please use the Applied Biosystems BigDye Terminator v1.1 & v3.1 5X Sequencing Buffer Calculator.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

The primer amount in the final reaction should be 3.2 pmoles. If the sequencing reaction is being diluted, the primer concentration may need additional optimization.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

We recommend using HPLC-purified primers for DNA sequencing. This will ensure the presence of full-length, highly purified primers that will minimize cycle sequencing noise and provides longer sequencing reads.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

If longer read lengths are needed, the BigDye Terminator v3.1 Cycle Sequencing kit has been formulated to deliver robust performance across a wide variety of DNA sequences while maximizing read lengths.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

The BigDye Terminator kit versions have different fluorescent dyes attached to the dideoxy terminators, and the dNTP/ddNTP ratio differs between the kits. The BigDye Terminator v1.1 Cycle Sequencing Kit is optimized for shorter fragments and for obtaining sequence close to the primer. The BigDye Terminator v3.1 Cycle Sequencing Kit is more suitable for larger templates and for obtaining longer read lengths.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Offscale data refers to data from samples that have signal intensities resulting in saturation of the CCD camera, on the capillary electrophoresis instruments. The maximum signal thresholds for raw data are 32,000 rfu for the Applied Biosystems 3730/3730xl DNA Analyzers, 3500/3500xL Genetic Analyzers, and SeqStudio Genetic Analyzer, and 8,000 rfu for the Applied Biosystems 310 Genetic Analyzer and Applied Biosystems 3130/3130xl Genetic Analyzers. For the SeqStudio Flex Genetic Analyzers, if the offscale recovery (OSR) is on, the maximum signal threshold is 65,000 rfu. If OSR is turned off, the maximum signal threshold for the SeqStudio Flex Genetic Analyzers is 32,000 rfu.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

The M13-tailed primers are used to simplify the workflow when sequencing PCR products and they reduce the loss of the 5' unresolvable bases. When the PCR primers contain M13 tails on their 5' ends, the M13 sequence is incorporated into the amplicons. This enables the use of sequencing master mixes containing either the universal M13 forward or M13 reverse primers. The sequence for the M13 forward and reverse primers are as follows:

-M13 forward primer sequence: 5′ TGTAAAACGACGGCCAGT 3′
-M13 reverse primer sequence: 5′ CAGGAAACAGCTATGACC 3′

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Primer Designer Tool is a free online tool to search for the appropriate PCR/Sanger primer pair from a database of >650,000 pre-designed primer pairs for resequencing the human exome. For more information, including a direct link to purchase the designed primers online go here (http://www.thermofisher.com/us/en/home/life-science/sequencing/sanger-sequencing/pre-designed-primers-pcr-sanger-sequencing.html?CID=primerdesigner).

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

We have the Primer Designer Tool, which is a free online tool to search for the appropriate PCR/Sanger primer pair from a database of >650,000 pre-designed primer pairs for resequencing the human exome. Go here (http://www.thermofisher.com/us/en/home/life-science/sequencing/sanger-sequencing/pre-designed-primers-pcr-sanger-sequencing.html?CID=primerdesigner) for more information, including a direct link to purchase the designed primers online.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

The pGEM control and M13 primer provided in the kit should be used for troubleshooting purposes. The pGEM is a control template that can be used to isolate issues with sample quality, thermal cycler, kit or sequencing reaction purification.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

We recommend a primer amount of 3.2 pmoles in the sequencing reaction.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

For long-term storage, the purified sequencing reaction should be dried and stored at -20 degrees C, protected from light.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

- After the sequencing reaction, post thermal cycling, the samples can be stored at 4 degrees C overnight or at -15 degrees C or -25 degrees C for long-term storage.
- After purifying with Centri-Sep or ethanol precipitation, dry down the samples, seal the plate with MicroAmp Clear Adhesive Film, and store, protected from light, at 4 degrees C for capillary electrophoresis (CE) preparation or at -20 degrees C until use.
- After BigDye Xterminator purification, to store for up to 10 days, seal the plate with MicroAmp Clear Adhesive Film, and store at 4 degrees C for CE preparation or at -20 degrees C. BDX plates can be stored at room temperature for up to 48 hours inclusive of time on the CE instrument.

For other DNA sequencing purification methods, please refer to the reagent-specific protocol.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Template quality impacts the DNA sequencing reaction. We recommended a spectrophotometer to determine DNA quality. Optimum absorbance ratios (A260/280) are between 1.8 and 2.0.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

RNA cannot be sequenced directly; instead we recommend transcribing the RNA to cDNA. The cDNA can then be used as a template for sequencing. For more information on recommended kits to convert RNA to cDNA go here (https://www.thermofisher.com/us/en/home/life-science/pcr/reverse-transcription.html.html?icid=fr-ssiii-main).

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

The Sanger sequencing reaction cannot be multiplexed, as the reaction should contain only one template and one primer. Multiple priming sites will result in sequencing noise and low quality data.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Our website provides many resources for obtaining information regarding sequencing applications. A few are listed below:

-Sanger Sequencing Overview (https://www.thermofisher.com/us/en/home/life-science/sequencing/sanger-sequencing/applications.html)
-DNA Sequencing by Capillary Electrophoresis Guide (http://tools.thermofisher.com/content/sfs/manuals/cms_041003.pdf)
-Seq It Out Video Series (https://www.thermofisher.com/us/en/home/life-science/sequencing/sequencing-education/seq-it-out.html)
-For additional resources, please see the Guides and Tools section on this page (http://www.thermofisher.com/us/en/home/technical-resources/technical-reference-library/capillary-electrophoresis-applications-support-center/sanger-sequencing-support.html).

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

DNA sequencing is the determination of the base-pair sequence of a DNA fragment by the formation of extension products of various lengths. DNA sequencing on capillary electrophoresis platforms is the process of reading nucleotide bases in a DNA molecule. During Sanger sequencing, DNA polymerases copy single-stranded DNA templates by adding nucleotides to a growing chain (extension product). This method involves copying single-stranded DNA with chemically altered bases called dideoxynucleotides which, when incorporated at the 3' end of the growing chain, terminate the chain selectively at A, C, G, or T. The terminated chains are then resolved by capillary electrophoresis.

Fragment analysis determines the relative size of a DNA fragment, typically a PCR product. One of the PCR primers is fluorescently labeled and the labeled PCR product is combined with a size standard to extrapolate the base-pair size of the sample.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.