dGTP BigDye™ Terminator v3.0 Ready Reaction Cycle Sequencing Kit

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.

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.