Applied Biosystems™ DS-30 Matrix Standard Kit (Dye Set D)

Catalog No.	Quantity
43-458-27	8 runs

Catalog No. 43-458-27 Supplier Applied Biosystems™ Supplier No. 4345827

Description

Includes

Contains 4 oligonucleotides labeled respectively with 6FAM™,HEX™,NED™,and ROX™

This Matrix Standard Set is used to generate the 'multicomponent matrix' required when analyzing fluorescent dye-labeled DNA fragments.

The DS-30 Matrix Standard kit contains 4 oligonucleotides respectively labeled with 6-FAM™, HEX™, NED™, and ROX™, pre-pooled in a single tube. This Matrix Standard Set is used to generate the “multicomponent matrix” required when analyzing fluorescent dye-labeled DNA fragments on the Applied Biosystems™ Systems. The Data Collection Software for these instruments uses the multicomponent matrix to automatically analyze the four different colored fluorescent dye-labeled samples in a single capillary.

Order Info

Each

Specifications

• Shipping Condition: Wet Ice
• For Use With (Equipment): SeqStudio 3500 and 3730xl
• Quantity: 8 runs

Frequently Asked Questions (FAQs)

What is a spectral calibration?

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.

Which fragment analysis matrix standards can I use for my Applied Biosystems 3130 Series instrument?

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.

I am using only one dye for fragment analysis but I see peaks in other colors below my peak of interest. Why is this?

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.

Can I switch dyes during a fragment analysis project?

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.

What is a dye set?

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.

Am I able to use dyes other than the recommended dyes for primer labeling prior to PCR amplification for fragment analysis?

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.

I would like to use another company's kit to generate fragments for my fragment analysis run. Is this possible?

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.

Do you have any fragment analysis-based kits for AFLP analysis?

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).

What kits are available for SNP genotyping by fragment analysis?

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.

Which fragment analysis applications can I run on the capillary electrophoresis (CE) instruments?

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).

What reagents do I need to start a fragment analysis project?

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).

When I started my run on the Applied Biosystems 3500/3500xL Genetic Analyzer, an error occurred: "Unstable Electrophoresis current detected, check for air bubbles" but I don't see any bubbles. What else can cause this?

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.

Do I need to replace the buffer every 14 days if the Applied Biosystems 3500/3500xL Genetic Analyzer is not in use?

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.

For Research Use Only. Not for use in diagnostic procedures.