Applied Biosystems™ 3730/3730xl DNA Analyzer Sequencing Standards, BigDye™ Terminator v3.1

Some of the causes of another sequence appearing under the primary sequence are:

• Contamination: There is more than one species of DNA present (e.g., multiple PCR products).

• Primers: Primer is annealing in more than one location on the template, primer dimer, primer degradation or not manufactured properly resulting in N+1 or N-1, carry over from PCR reaction, or primers pairs in a multiplex reaction that are not appropriate for multiplexing (i.e., primers anneal inappropriately).

• Spectral/Matrix: If the raw data signal intensity of the sample is too high or saturated it can exceed the amount of color bleedthrough (or spectral overlap) that the matrix (310) or spectral (3130, 3730, 3500) are removing, resulting in secondary peaks appearing in a very specific pattern (e.g., a red peak always appearing under a green peak). A change in the camera, laser, or optical alignment requires that a new matrix be made or a new spectral calibration be performed.

For more information on more than one sequence or set of peaks in a sequencing run, 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.

There is no fixed time as to when a spectral must be run. However, some users prefer to run a new spectral at a preset time to ensure it is up to date. Data quality will be the largest indicator of when a new spectral is required. A separate spectral calibration needs to be performed for each dye set, array type, and array length. For G5-RCT dye set users only: a spectral calibration must be re-run if the array detection cell is moved/repositioned. Refer to FAQ "When should I run a spectral calibration?" for more information on when a spectral should be run.

A new spectral calibration should be run:

- Whenever a new dye set is used on an instrument.
- After any optics adjustment performed by a service engineer.
- If the instrument has been moved.
- If you see a decrease in spectral separation (pull-up and/or pull-down peaks).
- If you alter any conditions (dye set, array type, or array length).
- For G5-RCT, Any4dye-HDR, and S dye set users only: Any time the array detection cell is moved/repositioned.

A spectral calibration is an algorithm applied to raw data, which converts it into the component 4 or 5 dye data stored in the sample files. A spectral is created for a specific dye set (combination of dyes), array type (4 or 16 capillaries), and array length (36cm or 50cm). It is used to correct for the natural overlap of the fluorescent dyes.

The sequencing standard has a shelf life of one year from the date of shipment, unless otherwise listed on the CofA. Our Terms and Conditions of sale can be found here(https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/Terms-and-Conditions-of-Sale.pdf).