missing translation for 'onlineSavingsMsg'
Learn More
  1. Home
  2. Products
  3. Biochemical Reagents
  4. DNA Reagents
  5. DNA Sequencing
  6. Applied Biosystems™ BigDye™ Terminator v3.1 Cycle Sequencing Kit

Applied Biosystems™ BigDye™ Terminator v3.1 Cycle Sequencing Kit

Product Code. 17152979 Shop All Applied Biosystems Products
Change view
Click to view available options
Quantity:
100 Reactions
1000 Reactions
24 Reactions
25,000 Reactions
5000 Reactions
Unit Size:
24 reactions
25000 reactions
Each
5 product options available for selection
Product selection table with 5 available options. Use arrow keys to navigate and Enter or Space to select.
Product Code. Quantity unitSize
17152979 25,000 Reactions 25000 reactions
15899786 24 Reactions 24 reactions
15781836 100 Reactions Each
15809796 1000 Reactions Each
15847581 5000 Reactions Each
Use arrow keys to navigate between rows. Press Enter or Space to select a product option. 5 options available.
5 options
This item is not returnable. View return policy

For Research Use Only. All usage must comply with product instructions.
Product Code. 17152979 Supplier Applied Biosystems™ Supplier No. 4337458

Please to purchase this item. Need a web account? Register with us today!

252789.64 EUR valid until 2026-06-30
BEST PRICE on Promo! Use promo code "28170" to get your promotional price.
This item is not returnable. View return policy

For Research Use Only. All usage must comply with product instructions.

Description

The 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.

The 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.

  • Improve the quality of your results for a wide range of sequencing applications
  • Optimized for long read lengths
  • Better dye mobility characteristics
  • Improved performance reading through GT-rich regions
  • Get longer, higher-quality reads with more uniform peak heights and optimal signal balance
  • Enhance your productivity and reduce costs

Get more uniform peak heights and improved accuracy

With BigDye™ Terminator v3.1 kit superior chemistry, you generate data with uniform peak heights and optimized signal balance. This results in longer, higher-quality reads and more accurate base assignments for heterozygote and mutation detection.

TRUSTED_SUSTAINABILITY

Specifications

Content And Storage • 10 x 20 mL tubes of BigDye™ Terminator v3.1 Ready Reaction Mix
• 4 tubes M13 (-21) Primer
• 4 tubes pREF-BDT™ Control DNA
• 1 x 233 mL tube of 5X Sequencing Buffer (may be stored at 4°C)

Store kit at -15°C to -25°C, and avoid repeated freeze-thaw cycles.
Format 12-well Strips, 8-well Strip, 96- or 384-well Plate
For Use With (Equipment) SeqStudio™ Genetic Analyzer, SeqStudio™ Flex Series Genetic Analyzer, 3500/3500xL Genetic Analyzer, 3730/3730xl DNA Analyzer Thermal Cyclers: GeneAmp™ PCR System 9700 Dual Series, Veriti™ Fast 96‐well and 384‐well Thermal Cyclers
For Use With (Application) Standard Sequencing
Product Line BigDye™ Terminator
Product Type Sequencing Buffer
Quantity 25,000 Reactions
Technique Fluorescent Dye Terminator Sequencing
Template Compatibility BAC DNA, Plasmid DNA (≤15Kb), PCR Amplicons, Single Stranded DNA

Frequently Asked Questions (FAQs)

What information should I include when submitting data to Genetic Analysis Technical Support?

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.

I have a long homopolymer stretch in my sample that I cannot get through. Do you have any tips for getting through it?

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.

I am trying to sequence GC-rich DNA without success. Do you have any tips for getting through this?

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.
I see another sequence running under mine. What can cause this?

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.
My data seems top heavy. It starts out strong and then gets weaker. What can cause this?

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.
I am seeing dye breakdown/artifacts in my data. What can cause this?

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.

I am getting shorter reads than I used to. What can cause this?

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.

My DNA sequencing data from the capillary electrophoresis system does not look good. Why?

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).

Can I dilute my BigDye Terminator reactions?

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.
What is a dye blob?

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.

What are the different ways to clean up my sequencing reactions?

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.

What is the difference between the regular BigDye (Terminator) kits and the BigDye Direct Cycle Sequencing Kit?

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).
I ran out of 5X Sequencing Buffer. What is the formulation so I can make my own?

The formulation of the 5X Sequencing Buffer that comes with the BigDye Terminator Kit is proprietary.
What is in the BigDye Terminator Ready Reaction Mix?

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.
Which kit is best for my application?

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.
My DNA is X bp. Which BigDye Terminator kit should I use?

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.
What is the difference between the BigDye Terminator v3.1 and v1.1 kits?

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.
What are the concentrations of the pREF-BDT and M13 primer controls that come with the BigDye Terminator kits?

The concentration of the pREF-BDT control is 200 ng/µL. The concentration of the M13 primer is 0.8 pmol/µL.
Should I use the BigDye Terminator v3.1 Cycle Sequencing Kit or BigDye Direct kit if I want to directly perform cycle sequencing on long-range amplicons (up to 10kb) using PCR primers for sequencing, instead of M13 primers?

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.
Do you offer the BigDye Terminator v1.1 & v3.1 5X Sequencing Buffer from the BigDye Terminator v3.1 Cycle Sequencing Kit as a standalone product?

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.

Can I purchase the pREF-BDT control from the BigDye Terminator kit separately?

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

What is the control in the BigDye Terminator kit?

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

For Research Use Only. Not for use in diagnostic procedures.

Product Title
Select an issue

By clicking Submit, you acknowledge that you may be contacted by Fisher Scientific in regards to the feedback you have provided in this form. We will not share your information for any other purposes. All contact information provided shall also be maintained in accordance with our Privacy Policy.