Fluorescently labeled fragments are produced via PCR when using dye-labelled primers specific to your marker(s) of interest. When resolved via capillary electrophoresis (CE) with an added size standard, the labeled fragments result in various profiles depending on the application and the sizes can be called in relation to the size standard. Using software specific to fragment analysis runs, one can use the data to analyse various genetic metrics. Allele frequency, Loss of Heterozygosity (LOH), and SNP genotyping are a few common applications of fragment analysis, but there are many uses for this quick and powerful assay.
- Standard Applications
- Microsatellite (STR) Analysis
- SNP Genotyping
- Allele frequency
- Standard Workflow
Isolate & Purify DNA
Various methods depending on your DNA source and application.
Dye-labeling and Fluorometric Detection such as the Qubit 2.0 Fluorometer enables accurate and target specific quantitation, even in the presence of buffers, salts and trace contaminants such as RNA or proteins. Spectrophotometry is also an option that is usually quite quick and cost-effective.
Dye-labeling and amplification of fragments in a thermal cycler. Cycling conditions and primers will vary depending on the application. If you need assistance designing your primers, please contact us.
Separation of fragments based on size. A size standard will be added to your sample (if you haven’t premixed your own). Please reserve the dye LIZ for the size standard. Your sample may contain up to 4 other dyes (see below),
Sizing and often genotyping. Please note that if you provide a size standard other than GS600LIZ, you must provide the fragment lengths of the size standard if also requesting that Retrogen perform the data analysis.
Standard Analysis at Retrogen
Send your purified, Dye-labelled and amplified PCR product for capillary electrophoresis at Retrogen. Retrogen uses Life Technologies’ dye set DS-33 enabling detection of up to 5-dyes per sample (Note: LIZ is reserved for the size standard). If you don’t have a size-standard included in your sample, Retrogen can provide GS600LIZ for accurate sizing of fragments from 20 to 600 bp.
Following CE, we will provide raw trace files from your runs, and upon request we can provide data analysis of your runs using GeneMapper software. Data will be sent as .csv files.
Fragment Analysis Software
Peak Scanner (Free)
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