DS-02 Matrix Standard Kit, for 3500/3730/SeqStudio™/SeqStudio™ Flex - FAQs

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.

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DS-02 (Dye Set E5), DS-30 (Dye Set D), DS-31 (Dye Set D), DS-32 (Dye Set F), and DS-33 (Dye Set G5) are all supported on the Applied Biosystems 3130 Series systems. Please refer to the Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide (http://tools.thermofisher.com/content/sfs/manuals/cms_041468.pdf) for more information.

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If the peaks in other colors are directly below the peak of interest, the issue could be that the fluorescent dye being used is not part of the selected dye set, the spectral calibration needs to be performed, or the peaks are offscale. Confirm that the dye set selected on the instrument is compatible with the dye being used, run a new spectral calibration if the correct dye set has been selected and, if the signal intensity is too high, decrease sample concentration during PCR or when preparing samples for electrophoresis.

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The SNaPshot Multiplex Kit is designed to interrogate up to ten single nucleotide polymorphisms (SNPs) at known locations on one to ten DNA templates in a single tube.

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Follow these recommendations for designing and evaluating primers:

- Primers included in a single reaction need to differ significantly in lengths in order to avoid overlap between the final SNaPshot products. A difference of 4-6 nucleotides between primer lengths is recommended as a starting point (5-7 nucleotides if running on POP-7 Polymer).
- The length of a primer can be modified by the addition of non-homologous polynucleotides at the 5′ end. Since the recommended annealing temperature for a SNaPshot control primer is 50 degrees C, the melting temperature for the complementary region between any primer and its corresponding template should be at least 50 degrees C.
- Poly (dT), poly (dA), poly (dC), and poly (dGACT) are 5′ non-homologous tails which are predicted to have minimal secondary structures. They have all been used successfully. Generally the signal patterns are not affected by the kinds of tails that are used. The 5′ poly (dT) tails however may interfere with the addition of 3′ ddA.
- The mobility of an oligonucleotide in capillary electrophoresis is determined by its size, nucleotide composition, and dye. Thus the effect of nucleotide composition on mobility can be significant when the primer is short. We strongly recommend that you test primers shorter than 36 nucleotides before being multiplexed to ensure that the final products are spatially resolved when analyzed on the instrument.
- Check primers for possible extendable hairpin structures within each primer and for extendable dimer formation between primers.
- HPLC purification of primers is recommended for oligonucleotides longer than 30 nucleotides. Heterogeneous primer mixtures containing mixed molecular weight oligonucleotides may yield undesired products that will confuse analysis.

For additional suggestions please refer to Appendix A of the SNaPshot Multiplex Kit manual (https://tools.thermofisher.com/content/sfs/manuals/cms_041203.pdf).

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The SNaPshot Multiplex Kit uses the E5 dye set which contains the dR110, dR6G, dTAMRA, and dROX dyes, and uses LIZ dye-labeled size standard.

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We do not recommend that you switch dyes in the middle of a project. Switching dyes may result in a shift in the allele sizes as dyes differ in their mobility. If dyes are switched in the middle of a project, it will be necessary to identify the size shift for all the markers/targets in the amplicon product range. The size shift will be consistent throughout the project and it is only necessary to make the adjustment once.

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A dye set is a combination of dyes that have minimum spectral overlap, which will allow you to multiplex products of similar size using different dyes. Although the size ranges may overlap, the use of a different dye will allow the software to easily distinguish between these products.

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We recommend using only Applied Biosystems dyes, as we provide spectral calibration reagents that have been optimized for our dye sets. Non-Applied Biosystems dyes have variable emission spectra and also require a spectral calibration generated for the specific dyes in use to correct for the spectral overlap between the dyes. You are responsible for obtaining the appropriate spectral calibration reagents and for optimizing custom dye sets to ensure that the dye labels do not affect PCR efficiency. The use of dyes outside of the recommended dye sets can result in pull-up and pull-down peaks, which may make allele-calling challenging.

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It is possible to use another company's kit. However, the vendor should be contacted to determine if they have protocol(s) for their kit(s) on the specific capillary electrophoresis (CE) instrument (specifically, the model you intend to use), and if they have reagents to run a spectral calibration or matrix run on the CE instrument with compatibility to the dyes that are part of the kit(s) in question.

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Unfortunately, we do not have fragment analysis-based kits for AFLP analysis. General information regarding AFLP can be found here (https://www.thermofisher.com/us/en/home/life-science/sequencing/fragment-analysis/amplified-fragment-length-polymorphism-aflp-analysis.html).

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We offer the SNaPshot Multiplex Kit for SNP genotyping. Please refer to our overview of SNP Genotyping by Fragment Analysis (https://www.thermofisher.com/us/en/home/life-science/sequencing/dna-sequencing/snp-genotyping-variant-detection-sequencing/snp-genotyping-fragment-analysis.html) for additional information.

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There are several fragment analysis applications that can be run on the CE instruments, such as: microsatellite analysis, SNP genotyping, fingerprinting, and relative fluorescence quantitation. Please see the DNA Fragment Analysis by Capillary Electrophoresis Guide for more details about these applications (http://tools.thermofisher.com/content/sfs/manuals/4474504.pdf).

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In order to determine the reagents needed, it will be necessary to gather initial information such as the following:
- What is the minimum and maximum amplicon size range?
- Will there be one target per sample or multiple targets (singleplex versus multiplex on the capillary electrophoresis instrument)?
- Is there overlap among the size of amplicons?

This initial information will help identify the spectral calibration needed for the capillary electrophoresis instrument, the fluorescent dye used to label the primer, and the size standard. Additional information can be found in the “Experimental Design” section of the DNA Fragment Analysis by Capillary Electrophoresis Guide (http://tools.thermofisher.com/content/sfs/manuals/4474504.pdf).

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Some of the other causes of an Unstable Electrophoresis current detected error message are:

1.Leak on the system
2. Polymer that:
-Has expired
-Has been left on the instrument for more than the recommended time
-Is a mixture of expired polymer and non-expired polymer
3. Running Buffer that was:
-On the instrument longer than 2 weeks
-Not filled to the fill line or evaporated below the fill line
4. An arcing event that was not cleaned afterwards using the water wash wizard
5. Not performing regular maintenance on the instrument
6. Hardware issues

Inspect the system for leaks. If you do not see any leaks on the system, perform the wash the pump chamber and channels wizard using the conditioning pouch and place fresh, non-expired polymer and fresh Anode and Cathode buffer containers. If the problem persists, a service call may be required.

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If the instrument is not in use, it is not necessary to replace the buffer every 14 days. However, the buffer level needs to remain at the fill line to prevent the capillary or array from drying out. Top off the buffer volume with water if necessary. Replace the anode and cathode buffer containers prior to starting a new run.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Instruments Support Center.