GeneScan™ 500XL ROX™ dye Size Standard - FAQs

If the larger peaks in the size standard are missing, it can also cause the sizing analysis to fail. This could be sample-related, where smaller ions and PCR products are preferentially injected into the capillaries and out-compete the injection of the larger products. To confirm, the size standard should be run without the sample.

If a size standard-only injection is performed, the larger size standard peaks may be missing due to slower migration of the sample. Slower migration can be due to some of the following:

-Degraded or expired polymer
- Old array or capillary
- Old or incorrect concentration of the buffer
- Old or expired HiDi Formamide
- Colder ambient temperature
- Oven temperature too low
If the issue persists, the instrument run time can be increased in order to allow for longer collection time of the largest size standard peaks. However, if the peak resolution is also broader than expected, this may suggest an instrument issue and a service call should be opened.

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

If a size standard-only run is performed and is missing the smaller fragments, degradation of the size standard may have occurred. Please check the expiration date either on the box or the Certificate of Analysis (COA). The size standard should be stored at 4-8 degrees C. Freezing of the size standard will cause loss of the smaller products and may also result in dye breakdown. The size standard should be replaced if expired or stored improperly. If the smaller peaks are missing, they may also be masked by the primer peak. The easiest fix is to go to the GeneMapper Manager, Size Standard tab, select the size standard definition you are working with and click the “Save As” button at the bottom. Rename the new standard and edit it by deleting any size smaller than 50 bp. Click OK, then Done and change the Size Standard in your project and re-analyze the data.

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

If the sample has strong signal and the size standard is weak, the sample may be preferentially injecting into the capillaries. The sample may have a high salt concentration and/or robust amplification. To confirm, a size standard-only sample should be run to confirm the performance of the size standard alone. If the signal intensity improves, the sample may require desalting or less PCR product should be used. If the size standard-only sample still shows low signal, the issue may be with possible degradation of the size standard and it may need to be replaced.

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

In GeneMapper Software, “failed sizing” is a result of the software being unable to identify the size standard peaks as defined by the selected size Standard. Please confirm that the size standard used in the sample matches the size standard selected in GeneMapper Software. The Size Match Editor function in GeneMapper Software allows you to view the size standard peaks sizing assignments. In the Size Match Editor window, the size standard peak heights can be reviewed, the user can determine if all the peaks in the size standard are present, or if there are any extraneous peaks in the same color as the size standard that are present. The Size Match Editor can be accessed by highlighting the failed samples in the Samples tab, and then going to the Analysis pull down menu > Size Match Editor.

-Within the GeneMapper Software's Analysis Method, the Peak Detector tab contains the minimum peak height setting for each color (blue, green, yellow, red, purple, and orange). If the size standard peaks are below the minimum peak height setting on the Y-axis, typically 50, the software will ignore these peaks (this threshold is different for fragment analysis that occurs in the 3500 Data Collection software). Although this setting may be altered, it may be necessary to troubleshoot further before doing so. If the sample has strong signal and the size standard is weak, the sample may be preferentially injecting into the capillaries. The sample may have a high salt concentration and/or robust amplification. To confirm, a size standard-only sample should be run to confirm the performance of the size standard alone. If the signal intensity of the size standard improves, the sample may require desalting or less PCR product should be used. If the size standard-only sample still shows low signal, the issue may be possible degradation of the size standard or the Hi-Di Formamide and they may need to be replaced. It may also be a sign that the capillary array is old or clogged.
-If there are any extraneous peaks, this can be due to pull-up peaks from overloaded samples. Extraneous peaks in the same color as the size standard dye label may interfere with the sizing algorithm in the analysis software. The sample can be reduced to prevent the pull-up peak from overloaded samples. If the sample is not overloaded, please confirm that the fluorescent dye in use is part of the dye set selected. The spectral calibration may also need to be re-run.
- If the larger peaks in the size standard are missing, it can also cause the sizing analysis to fail. This could be sample-related where smaller ions and PCR products are preferentially injected into the capillaries and out-compete the injection of the larger products. To confirm, the size standard should be run without the sample.
- If a size standard-only injection is performed, the larger size standard peaks may be missing due to slower migration of the sample. Slower migration can be due to some of the following:
1.Degraded or expired polymer
2.Old array or capillary
3.Old or incorrect concentration of the buffer
4.Old or expired HiDi Formamide
5.Colder ambient temperature
6.Oven temperature too low
If the issue persists, the instrument run time can be increased in order to allow for longer collection time of the largest size standard peaks. However, if the peak resolution is also broader than expected, this may suggest an instrument issue and a service call should be opened.

- If the smaller peaks in the size standard are missing, they may be masked by the primer peak. The easiest fix is to go to the GeneMapper Manager, Size Standard tab, select the size standard definition you are working with and click the “Save As” button at the bottom. Rename the new standard and edit it by deleting any size smaller than 50 bp. Click OK, then Done and change the size standard in your project and re-analyze the data.

The Size Match Editor will allow overriding of the sample quality but it is important to confirm the size standard peaks are correctly identified. Otherwise, the peak sizes of the PCR products may be shifted. To override the sizing quality in the Size Match Editor, click on the “Override SQ” button on the top, middle of the window.

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

- As the size of a fragment is calculated based on the size standard with which it co-migrates, dye-labeled DNA fragments can yield different sizes when run with a different instrument, polymer, capillary array length, or size standard.
- Check to see whether the polymer, buffer, or array has expired and/or is degraded. If so, they will need to be replaced.
- Check that the analysis parameters, analysis method, and size standard selected in the analysis software are the same.
- It is also possible to observe slight shifts in sizing if the lab temperatures differ. This will also impact the migration of the sample.
- If multiple injections of the control yield the same size, then it may be necessary to note the offset in sizes for a given fragment as this will confirm the instrument precision.

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

If the fragments have shifted in size and the same capillary electrophoresis (CE) instrument, dyes, and run module are being used, the polymer should be checked to see how long it has been on the instrument and whether it has expired. The polymer can be kept on most CE instruments for 7 days, with the exception of the 3500/3500xL Genetic Analyzers where the polymer may be on the instrument for up to 14 days if the lab temperature is below 25 degrees C.

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

The size of the PCR product may not match the size confirmed by sequencing because a size standard is used to extrapolate the base-pair sizes of the sample product peaks. The fragment analysis software uses the size standard in each sample to create a standard curve for each sample. It then determines the relative size of each dye-labeled fragment in the sample by comparing fragments with the standard curve for that specific sample. The mobility of the fragments is affected by the sequence composition, the fluorescent label, and electrophoresis conditions. Although the PCR product size may not match that confirmed by sequencing, once the size has been established, the precision, or reproducibility, will be consistent for a given fragment.

If the sequence composition, fluorescent label, electrophoresis conditions, or size standard are changed, this will impact the relative size of the fragment.

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

For data run on capillary electrophoresis platforms, the troubleshooting process consists of the following steps:

- Identify the problem, i.e., low signal intensity, broad peaks, noisy data etc.
- Check for proper storage of reagents and expiry dates.
- Run the controls. For fragment analysis applications, running the installation standard or size standard on its own can help direct troubleshooting efforts. A possible troubleshooting procedure would be:

1. Mix 12.5 µL of HiDi Formamide and 0.5 µL of Internal Size Standard (i.e., LIZ 600 Dye Size Standard, ROX 500 Dye Size Standard) per well/capillary (for example, A 16 capillary array would need 16 wells, each containing 12.5 µL HiDi Formamide and 0.5 µL of Internal Size Standard).

2. Run the standards using Standard Run Modules. Sizing should pass in the Analysis Software using a Default Analysis Method and Size Standard Definition.

This can be used to determine if the size standards, instrument hardware, and consumables on the instrument (i.e., polymer, buffer, capillary/array) are working properly and if the proper software settings have been selected for the analysis. If the standards do not look good and weekly maintenance has not been performed, perform the weekly maintenance and re-run the standards. If the standards still do not look good, contact Technical Support at techsupport@thermofisher.com. If the installation/size standard plate looks good, go to step 3:

3. Set up a plate of PCR reactions using a laboratory internal DNA control sample or your QC DNA, as per your laboratory's established protocol.

4. After the PCR reaction is complete, dilute the sample as per your laboratory protocol and mix 1 µL of diluted sample, 0.5 µL Internal Size Standard, and 10.5 µL HiDi Formamide. Alternatively, the diluted sample can be added to the size standard-only plate prepared in step 1, to save on reagents.

5. Denature the sample at 95 degrees C for 3 minutes, and place on ice for 3 minutes.

6. Run the samples using Standard Run Modules.

This can help determine if the problem is with the chemistry, thermal cycler, template, or the primers. Running the samples on an agarose gel can sometimes help in further defining areas to troubleshoot. If the standards and internal controls all look good but you experience data quality issues with your samples, the problem is most likely in the template or primers being used for that sample. Please contact Technical Support at techsupport@thermofisher.com for further assistance.

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

Switching size standards in the middle of a project may result in a shift in the allele sizes, as size standard dyes differ in their mobility. If the size standard change is done, it will be necessary to identify the size shift for all the markers/targets in the product.

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

Switching size standards in the middle of a project may result in a shift in the allele sizes, as size standard dyes differ in their mobility. If the size standard change is done, it will be necessary to identify the size shift for all the markers/targets in the product. Switching from a LIZ dye to a ROX dye will also require running it under a new dye set. Make sure the dyes you are using are compatible with the dye set D and that you have a spectral for the new dye set.

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

The GeneScan 500 LIZ Dye Size Standard and GeneScan 500 ROX Dye Size Standard differ in the fluorescent dyes. The GeneScan 500 LIZ Dye Size Standard is used with the 5-dye chemistry and the G5 dye set. The GeneScan 500 ROX Dye Size Standard is used with the 4-dye chemistry and the DS-30, DS-31 and DS-32 dye sets.

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

The size standard expiration date can be found either on the original box/pouch or the Certificate of Analysis (COA). To access the COA, click here and search with the Lot No. If there is no expiration date listed on the box or COA, the warranty date is 1 year from date of shipment.

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

Size standards stored at -20 degrees C can result in loss of the smaller size standard peaks, lower signal, or no signal. The size standard should be run with HiDi-Formamide only to determine the impact to the size standard peak intensity.

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

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.

Unfortunately, we do not have fragment analysis-based kits for AFLP analysis. General information regarding AFLP can be found here (https://www.thermofisher.com/us/en/home/life-science/sequencing/fragment-analysis/amplified-fragment-length-polymorphism-aflp-analysis.html).

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

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.

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.

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.