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Custom 5' Fluorescent Labeled/Unlabeled Pairs, 80,000 picomoles⁄each - FAQs

View additional product information for Custom 5' Fluorescent Labeled/Unlabeled Pairs - FAQs (4304979, 450062, 4304981, 450059, 4304982, 4304976, 4304977, 450056, 4304978)

16 product FAQs found

I am performing PCR amplification prior to fragment analysis and my PCR reaction is failing. Why?

If the PCR products are run on an agarose gel and there are no bands, see here for possible reasons. If the PCR products can be visualized on an agarose gel but not on the capillary electrophoresis (CE) instrument, and the size standard is present in the sample file, the issue may be with the fluorescently-labeled primer. Re-synthesizing the primer would be recommended.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

If I use a primer labeled with dye X, I get very little or no signal. But if I use the same primer labeled with dye Y, it works well. Why is this?

Fluorescent dyes can differ in their emission spectrum, excitation spectrum, and excitation efficiency and, as a result, the intensity of the emitted fluorescence is different for each dye and, therefore, optimization of the sample concentration needs to be performed to account for the differences in the dye signal strength. For instance, in the G5 dye set, the signal intensities from highest to lowest are 6-FAM, VIC, NED, and then PET. If the same primer sequences are labeled with 6-FAM and NED, more of the NED-labeled sample must be loaded on the instrument to have signal comparable to the same 6-FAM-labeled sample. For a list of relative signal intensities, please see the Dyes section in the DNA Fragment Analysis by Capillary Electrophoresis Guide (http://tools.thermofisher.com/content/sfs/manuals/4474504.pdf).

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

My PCR products are above 100 bp. What is the high signal I am observing on the capillary electrophoresis (CE) instrument before the 50 bp fragment?

The signal before the 50 bp fragment is most likely excess fluorescently labeled primers or primer dimers. The extraneous peaks may not interfere with the allele calls if the PCR products are above 100 bp.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

I am able to visualize PCR products on an agarose gel but not on the capillary electrophoresis (CE) instrument. Why?

If the PCR products can be visualized on an agarose gel but not on the CE instrument, and the size standard is present in the sample file, the issue may be with the fluorescently labeled primer. Re-synthesizing the primer would be recommended.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

What primer purity do I need for fragment analysis applications?

For the majority of fragment analysis applications, desalted primers are fine. For SNaPshot primers, HPLC-purification will be necessary.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

How should I store fluorescently-labeled oligos?

Fluorescently-labeled oligos should be protected from light to prevent photobleaching. They should be wrapped in aluminum foil or kept in a cardboard box in a -20 degrees C freezer.
To minimize freeze/thaw cycles, we recommend you aliquot the stock dilution into small “working” volumes, according to the number of reactions, and store at -20 degrees C. Prior to use, the solution should be lightly vortexed and spun down using a bench-top centrifuge.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

What is the shelf-life of the oligos?

The oligos are good for one year from the date of shipment when stored at -20 degrees C.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

At what concentration should I resuspend my oligos for use in PCR prior to fragment analysis?

A stock concentration can be between 10 µM and 100 µM. A working concentration can then be made depending on the concentration needed in the PCR reaction.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

How should I resuspend my oligos for use in PCR prior to fragment analysis?

Primers can be reconstituted in sterile 1X TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0), sterile 1X low TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0), or molecular-grade, nuclease-free water. For more information on how to dilute your custom primers, refer to the following document (http://tools.thermofisher.com/content/sfs/brochures/cms_043004.pdf).

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Do I have to optimize my PCR reaction with the labeled primers if the reaction worked with the unlabeled primers?

The use of labeled primers does not typically require the need for further optimization of the PCR reaction. Some optimization may be needed regarding dilution of the PCR product on the capillary electrophoresis instrument to prevent overloading of the capillaries and saturation of signal on the CCD camera. Please refer to Chapter 4 of the DNA Fragment Analysis by Capillary Electrophoresis Guide (http://tools.thermofisher.com/content/sfs/manuals/4474504.pdf) for additional recommendation on sample loading concentration and optimizing signal intensity.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

How many PCR products can be multiplexed prior to loading on a capillary electrophoresis instrument?

The number of PCR products that can be pooled is determined by the size range of the PCR products and whether 2, 3 or 4 dyes in the same dye set are used for labeling the primers. PCR product sizes may overlap, but it is necessary to use different fluorescent dyes so they can be distinguished from each other.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Can I multiplex my PCR reaction for loading onto a capillary electrophoresis instrument?

It is possible to amplify multiple targets within the same PCR reaction. However, there are limitations such as primer-oligomer formation, loss of specificity, and decreased yield of specific products which will require a significant amount of optimization to overcome. We recommend that you perform the PCR reactions in singleplex and then pool the PCR products prior to loading on the capillary electrophoresis instrument.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

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).

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

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).

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.