Thermo Scientific appliedbiosystems SeqStudio Flex Series - During a Run; Results; Materials for Routine Operation

During a run
During a run, the instrument:
•
Prepares the capillaries by pumping fresh polymer under high pressure from the polymer delivery
pump to the waste position in the cathode buer container (CBC).
•
Electrokinetically injects the sample into the capillaries by briefly applying a low voltage.
•
Washes the capillary tips in the rinse position of the CBC, then returns the capillary to the buer
position of the CBC.
•
Ramps the voltage up to a constant level.
A high electric field is created between the ground end of the anode buer container (ABC) and
the negative voltage applied to the load header of the capillary array. This field pulls the negatively
charged DNA through the separation polymer. The smaller fragments migrate faster than the larger
fragments and reach the detector first.
To ensure optimal separation and maintain denaturation of the DNA, the capillaries are thermally
controlled in the oven and in the detection cell. The oven has a Peltier heat unit and fan-circulated
air.
In the detection cell, the dyes attached to DNA are excited by a narrow beam of laser light. The
laser light is directed into the plane of the capillaries from both the bottom and top. A small amount
of laser light is absorbed by the dyes and emitted as longer wavelength light in all directions.
•
Captures the fluorescent light on the instrument optics while blocking the laser light. The light
passes through a transmission grating, which spreads the light out. The light is imaged onto a
cooled CCD array. For each capillary, 20 zones on the CCD are collected to provide 20-color data
for each capillary.
•
Converts the 20-color data into multi-dye data for the entire run. For sequencing applications,
4 dierent dyes are used to determine the 4 bases A, G, C and T. For fragment analysis
applications, up to 6 dyes can be used to determine fragment sizes in a single run.
Results
The software generates an electropherogram (intensity plot) for each dye based on the migration of
DNA fragments over the run and generates primary analysis results:
•
For sequencing applications, the electropherogram is adjusted to compensate for slight mobility
dierences due to the dyes, then basecalling is performed and quality values are assigned. Results
are generated in AB1 file format.
•
For fragment analysis, the software uses the internal size standard to assign a fragment size to
each peak and a sizing quality value to the sample. Results are generated in FSA file format.
Materials for routine operation
Materials for limited operation are provided when the instrument is installed. These materials are listed
in “Instrument consumables handling, usage limits, and expiration” on page 25. For more information
on ordering the materials for routine operation, you can do either of the following:
•
See Appendix D, “Catalog numbers”
•
Contact your local representative
Chapter 1 Product information
Materials for routine operation
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SeqStudio
™
Flex Series Genetic Analyzer with Instrument Software v1.0 User Guide