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View additional product information for BigDye™ Terminator v3.1 Cycle Sequencing Kit - FAQs (4337457, 4337456, 4337455, 4337454, 4337458)
22 product FAQs found
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.
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 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.