Fragment Analysis (FA), also known as Fragment Length analysis (FLA), is a powerful technique used in a wide-range of applications—detection of mutations, genotyping, identifying short tandem repeats, and DNA profiling. Eurofins Genomics offers highest accuracy in sequencing the fully prepared samples via capillary electrophoresis using ABI's 3730xl DNA analyzers. Our current offering in Fragment Analysis includes the Ready2Load service.
Fragment Analysis - Ready2Load Service
The Fragment Analysis - Ready2Load service allows you to get your assay samples analyzed on our ABI DNA analyzers.
What you do
- Design your fragment analysis assay using the dyes from DS-33 (see below)
- Perform PCR based on your assay design
- Assemble your sample(s) (dye-labeled PCR Product + DNA Size-standards + Hi-Di Formamide)
- Ship the assembled samples to Eurofins Genomics in a secure 96-well microtiter plate (see below)
What we do
- Load and run the samples on ABI 3730xl DNA analyzer
- Send the sequence data to your email
Please note that we currently accept samples from assays using the following dye combination.
Applied Biosystems 3730xl Compatible Fluorescent Dyes and Standard
|DNA size standard
|Fluorescent Dye Label
Example of an Applied Biosystems 3730xl Compatible 96-well plate
||MicroAmp Optical 96-Well Reaction Plate (0.1 mL) or equivalent product
Ensure that the 96-well plates are sealed securely before shipping the same. Please adhere to the guidelines in the Sample Submission Guidelines for Fragment Analysis.
Fragment Analysis - Ready2Load Services
When all that is remaining to complete your Fragment Analysis assay is for the samples to be run on a ABI 3730xl DNA Analyzer, ship your sample to Eurofins Genomics! The Ready2Load Fragment Analysis service offered by Eurofins Genomics includes the following services:
- Accurate separation of fragments by Capillary Electrophoresis (CE) on ABI 3730xl sequencing machine under standard conditions
- Sequence data sent via email as .fsa files (use GeneMapper or GenoTyper analysis software to read the same).
- Typical turn around time for the delivery of .fsa files is within 3 days.
- Analysis of fragments spanning longer lengths will be considered on request
About Fragment Analysis:
Dye-Labeled DNA fragments can be analyzed on the Applied Biosystems 3730xl DNA analyzers. PCR amplification of a Dye-labeled primer is the most widely used method for incorporating dyes in DNA fragments.
Several genetic analysis experiments incorporate the dye in DNA fragments to gather critical information about the organisms. The applications of fragment analysis that we can process include include SNP Genotyping, Amplified Fragment Length Polymorphism (AFLP), loss of heterozygosity and INDEL evaluations.
Please note that we currently only accept samples labeled with the dyes in the DS-33 Matrix Standard Kit (Dye Set G5), i.e., 6-FAM, VIC, NED, PET and LIZ. The 600 LIZ® DNA Size Standard must be added to you Ready2Load samples. Please adhere to the guidelines in the Sample Submission Guidelines document.
|Fragment Analysis - Ready2Load
|Up to 5 96-well plates
||3 business days
|Greater than 5 96-well plates
||4 business days /each set of 5 plates
Sample Submission Guidelines for Fragment Analysis
Eurofins Genomics (US) offers the ‘Fragment Analysis – Ready2Load’ service using ABI 3730xl DNA analyzers. Our workflow is optimized to offer the service for the PCR products generated by microsatellite, SNP, LOH, AFLP, RFLP, T-RFLP, ARISA or other protocols.
Only the following dye combinations can be used as matrix and for labelling the PCR products:
ABI Standard Dye Set DS-33 - 6-FAM, VIC, NED, PET and LIZ*
*the dye LIZ is typically reserved for the size standard and should not be used for PCR product labelling.
The protocol for the Ready2Load fragment analysis at Eurofins Genomics US is optimized for analysis of DNA fragments 50–600 bp. The LIZ 600 size standard must be included in your samples for fragment analysis.
Sample preparation for ‘Fragment Analysis – Ready2Load’ Service
Please adhere to the following guidelines, to assemble samples for Fragment Analysis – Ready2Load service.
- Run PCR using dye labeled primers that can be analyzed with ABI Standard DS-33 filter set
- Prepare solutions of PCR products at 50–100 ng/μL
- Assemble the samples for ‘Fragment Analysis – Ready2Load’ Service, by mixing the following:
- Hi-Di Formamide loading buffer, 10 μL (Applied Biosystems)
- Dye-Labeled internal size standard, 0.5 μL (use only LIZ-600).
- PCR product, 2 μL
- The total sample volume should be 10–15 μL.
Sample Submission formats
Please note that you can submit your samples in one of the following two formats:
- 1.75 mL centrifuge tubes
- 96-well plates (MicroAmp® Optical 96-Well Reaction Plate, ThermoFisher, catalogue no. 4316813, or equivalent)
Ship the liquid samples at ambient temperature to:
12701 Plantside Drive,
Louisville, KY 40299
- Use 8-cap strips for sealing 96-well plates (recommended)
- Seal the plate tightly and avoid folding; don’t freeze and thaw the samples after sealing.
To place an order for ‘Fragment Analysis – Ready2Load’ service, you can submit the details of your assay samples and payment information in an excel file via email to GenomicsSupport@eurofins.com. Additional details are included below
- Download the excel order form from https://www.eurofinsgenomics.com/en/products/dna-sequencing/fragment-analysis/
- Send the filled order form electronically to genomicsSupport@eurofins.com and as a hard copy together with the samples
- Label your samples according to the sample names indicated in your sample list:
- Single tubes – on the lid
- 96-well plates – on the plate frame
- The data will be delivered in .fsa format within 3 days after receiving the samples
- Results can be viewed using the following free/paid softwares
Please note that other softwares can also be used to view .fsa files. These include GeneMarker (SoftGenetics), Peak Studio (http://fodorlab.uncc.edu/software/peakstudio), Xplorer (http://www.dnatools.com/download.html), STRand (http://www.vgl.ucdavis.edu/informatics/strand.php), DAx (http://www.dax.nl/dax.htm)
- GeneMapper (Applied Biosystems) Software
- Peak Scanner (Applied Biosystems)
A note about sample concentration
- Concentration across all samples submitted in an order must be normalized
- For multiplex analysis, the concentrations of the PCR products in a sample must be normalized in order to get a balanced profile (recommended variations in concentration <50%)
Please do not hesitate to contact us in case of any questions! Email: GenomicsSupport@eurofins.com Phone: 1-800-688-2248