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View additional product information for BigDye™ Terminator v1.1 Cycle Sequencing Kit - FAQs (4337449, 4337452, 4337451, 4337450)
27 product FAQs found
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 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.
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 DNA sequence immediately after the primer is needed, the BigDye Terminator v1.1 Cycle Sequencing Kit has been designed to deliver optimal 5' resolution and basecalling in shorter fragments when used in combination with POP-6 polymer and a 50 cm array.
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.