Our 3730xl 96-Capillary Genetic Analyzer is calibrated for several dyes sets. One dye in each set is reserved for an internal lane size standard, the other dyes are used to label the fragments (typically a modification added to the forward primer on the 5’ end).
|Promega Power Plex*||DS-30||DS-32||DS-33|
|CXR size standard||ROX size standard||ROX size standard||PET|
|LIZ size standard|
*used for Cell Line Authentication with the GenePrint® 10 System and PowerPlex® Systems
Frequently Asked Questions:
Do samples need to be purified before submitting?
No, unlike sequencing samples Fragment Analysis samples do not require purification before submission, although this can improve the peak morphology for some loci.
What size standard should I use?
First and foremost, the dyes used to label fragments is the most import factor to consider when choosing a size standard. Second is the size of the products being amplified. Third, the need for multiplexing to reduce overall costs. Liz size standard is labeled with a fifth dye and allows users to multiplex a greater numbers of dye labeled fragments in a given lane; compared to the four dye system available with ROX. Both LIZ and ROX are available in a number of upper fragment limit size options.
What size standards do we offer?
If you’re starting a new project, or running a new locus under 500 nucleotides in length, please use our new size standards Order form Fragment Analysis-New Rox or Liz (GelCo.):
- Gel Company-Liz-500–contains 16 single-stranded fragments, sizes: 35, 50, 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490 and 500.
- Gel Company-Rox-500–contains 25 single-stranded labeled fragment of: 70, 80, 90, 100, 120, 140, 160, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 425, 450, 475, 490, and 500.
If you’re continuing an older project and data will be combined, or have fragments between 500-600nt use the following order form Fragment Analysis-Old Rox or Liz (ABI) for the following size standards:
- Genescan Liz-500–contains 16 single-stranded fragments, sizes: 35, 50, 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490 and 500.
- Genescan Liz-600–contains 36 fragments that are labeled with the 5th dye LIZ®. The size standard peaks are 20, 40, 60, 80, 100, 114, 120, 140, 160, 180, 200, 214, 220, 240, 250, 260, 280, 300, 314, 320, 340, 360, 380, 400, 414, 420, 440, 460, 480, 500, 514, 520, 540, 560, 580 and 600 bps. Note: we only offer this on an as needed basis, for projects with fragments above 500 and for long term projects that require it for combining with previous data.
- Genescan Rox-500–contains 16 single-stranded labeled fragment of: 35, 50, 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490, and 500 bases.
Am I committed to using same size standard for the WHOLE project for one locus?
The dyes used to label the single stranded fragments in the size standards have specific molecular weights, which affects migration rates of your samples through the capillaries. The number of fragments multi-plexed can affect migration rates as well. The same sample run with different size standards WILL migrate at a different speed, resulting in a different fragment size/allele call.
How do I order labeled primers?
Interested in saving money? Investigate this economic method.
“An economic method for the Fluorescent labeling of PCR fragments” (pdf link) for a method that can save money by simply labeling the Forward Primer with an M13-tail, then incorporating an M13 oligo with a dye attached into the PCR reaction. Our research group has used this approach for over a decade and has produced good results. We have found in our years of experience, (for what it’s worth we’ve overseen many projects with many organisms), the primers design and more specifically the locus being amplified, determine the quality of results, rather then the method of labeling the primer. People who may criticize this technique do so because their primers were not designed well to begin with and the locus being amplified is problematic.
Can I order labeled primers with dyes other then the ones listed?
Yes, as long as the dye being ordered has nearly the same emission spectra, but be CAREFUL. Substituting VIC with a less expensive HEX, will result in the exclusion of NED, because HEX has a slightly higher emission which causes HEX to have spectral overlap with NED.
So if HEX and NED are run together with the DS-33 filter the NED signal is contaminated with signal from HEX. This is called “spectral bleeding” or “pull-up”. This COULD make calling alleles a problem. Use caution with your experiments.
Can I run labeled primers in combinations not listed above?
Technically no, unless we create a custom dye set for your dyes. This is possible, but will require some work on your part. Contact us for more information. It’s best to stick to the combinations listed.
Is there a limit to the number of loci run in each lane?
Multiple loci may be run in the same lane/well if they are separated either by size or color. The total number of loci run together depends on many factors. Typically up to 4 is fine. While we do see users running more then 4, this all depends on the loci and more specifically the primer being used. The PCR amplicons also present very different motifs in the alleles, which can be problematic if not optimized. We can discuss this more on an individual basis.
How can I improve the peak morphology of my amplified products?
A Touch Down (TD) Cycling Protocol can help improve signals for some loci. When starting a new project we recommend comparing a Standard single annealing temperature protocol with a Touch Down protocol. What you might see is a Touch Down will drastically reduce the number of stutter bands produced, other times it has no affect. We also recommend trying at least 2 taq’s. Qiagen Type-It taq has some kind of magic fairy dust in it, (probably just table salt) and works wonders for some templates, while for others it’s not worth the extra cost for the secret ingredients.
Other Information about Microsatellites
Protocol’s for building an Enriched Microsatellite Library for Novel Species
Glenn, T.C. and N.A. Schable. 2005. Isolating Microsatellite DNA loci. Methods in Enzymology, 395:202-222.
Glenn, T.C. and N.A. Schable. Updated Protocol. Isolating Microsatellite DNA loci.
Other Information about AFLP’s
S. Trybush, et al. Getting the most out of fluorescent amplified fragment length polymorphism. Canadian Journal of Botany. 2006. 84:1347-1354
Springer protocols: Amplified Fragment Length Polymorphism Analysis of Salmonella enterica