Applied Biosystems™ dGTP BigDye™ Terminator v1.0 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.

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.

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.

The dGTP BigDye Terminator Cycle Sequencing Kit can be run on all of our capillary electrophoresis platforms:

- Applied Biosystems 3500/3500xL Genetic Analyzers
- Applied Biosystems 3730/3730xl DNA Analyzers
- Applied Biosystems 310 Genetic Analyzer
- Applied Biosystems 3130/3130xl DNA Analyzers

However, due to the nature of the chemistry, compressions in the data are expected.

The dGTP BigDye Terminator Cycle Sequencing Kit should be used for difficult templates, such as templates that are GT-rich, G-rich templates or are difficult to sequence with other kits. They should be used only when you cannot obtain good data using standard BigDye Terminator kits.