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).
Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.
Can I purchase the -21 M13 forward control primer from the BigDye Terminator kit separately?
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.
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.
Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.
