Fragment Analysis Information
Our facility has the ability to carry out fluorescent DNA fragment analysis--this can include analyzing labeled products generated by AFLP, STR, or even DNAse footprinting or primer extension. We have the most experience using the Genemarker software package from SoftGenetics. Because of the different types of experimental manipulations and output that fall under the heading "fragment analysis," we have options for sample preparation, drop off, and computer analysis.
The Fragment Analysis service on the 3730 is provided per 96 well plate only.
Please note these are internal prices, external users have a surcharge.
User provides at least 10-15ul of each cleaned up PCR products in either 0.2ml arrayed striptubes or plate. Molecular weight ladder (please inquire which ladders are available) with formamide will be added to 2ul sample.
RTG Genescan Plate
User provides half skirted, foil sealed plate (we provide plate and seal) containing appropriately aliquoted amounts of molecular weight ladder and sample in high quality deionized formamide and a minimum volume of 12ul. Please note the dye set to use on the submission form.
- Sample Naming. If unique sample names are to be used, an Excel sample sheet containing the names needs to be uploaded to our server--we can show you how to do this if necessary. The default names will be 1 to 96 with well positions (01A, 01B, etc).
- Dye Set. Normally if the ladder is Rox, then we use dye set D. If the ladder is Liz, then dye set G5 is needed. Please note the dye set to use on the submission form.
- Maximum Fragment Size. Sample run time by default is limited to fragments of about 600 bp or below, but we can go higher upon consultation. This is due to both labeled standard and software limitations.
- No dried samples. No samples covered in oil.
- Controls. We strongly recommend you include at least one sample representing a negative control and one well representing a positive control. That will help insure that we've loaded your samples correctly, and that your PCR reaction was successful.
Generation of labeled fragments
Our ABi instrumentation is spectrally calibrated to detect a number of fluorophores. The 3730 can run either filter set D or set G5 samples per plate. These include the dyes 6-FAM (blue), HEX (green), NED or TAMRA (yellow), and ROX (red) for filter set D. ROX is reserved for the internal MW standard run in every sample on filter set D. The G5 filter set allows for four dyes attached to samples, using LIZ (orange) as the internal lane standard. The dyes used for samples in G5 are: FAM, HEX, NED or Tamra, and PET. The DNA Synthesis core is experienced making custom primers with fluorophores, such as FAM, TAMRA and Hex.
Sample Setup for Fragment Analysis
For RTG Genescan the following steps also need to be taken before submitting samples. The typical reaction setup is 2ul cleaned up sample with 0.3ul MW ladder with the reaction formamide brought up to a volume of 12ul.
A majority of the customers use Geneflo Rox-625 as the internal ladder. There are a variety of size ranges of ladder from various companies. All Liz ladders are from ABi/Life Technology. As per the section on generating labeled products, MW ladder size impacts which dye set to use for the run and for how long the run is. By default the run is for Rox 625bp, but if the read needs to go further or use a different dye set, please note these on the submission form.
A critical factor is the formamide used for sample setup- NOT ALL COMMERCIAL FORMAMIDES WILL WORK! We use the ABi Hi-Di formamide; though deionized formamide from Sigma also works. If you want to use another brand of formamide, contact us first.
Here's where it gets interesting! In our opinion, and of many in the fragment analysis community, analysis software options are still limiting. Currently the data generated is an fsa file from our Applied Biosystems 3730. There are a couple of options to help you analyze your data.
GeneMapper and GeneMarker are the standard programs available. These are quality Windows based programs with many features. If fragment analysis is going to be a regular service request, spend the money on the software. It will save significant time interpreting data. Peak Scanner, STRand and Genographer are free but each has its own limitation.
If there are questions please contact us at email@example.com.