Troubleshooting Late Migration on the Agilent 2100 Bioanalyzer System

Answer

This Information Applies To: 2100 Bioanalyzer system, 2100 Expert software

Issue:

How to identify and troubleshoot late migration of the ladder, marker, and sample peaks in data generated on the Agilent 2100 Bioanalyzer system.

Background

The ladder, marker, and sample peaks in the affected run take considerably longer to migrate compared to the peaks in the demo file of the same assay type. Late migration is usually accompanied by errors, similar to the ones shown in Figure 1. These errors indicate that the marker and ladder peaks are not detected after the anticipated number of seconds or not detected at all.

 
 

Figure 1. Errors in the Agilent 2100 Expert software, caused by late migration

Verifying Late Migration:

• Open the data file with suspected delayed migration and the demo file for the assay. Demo files saved in the following location C:\Program Files (x86)\Agilent\2100 bioanalyzer\2100 expert\data\samples\demo\electrophoresis.
• Turn off the analysis for both the files by clicking the blue stop button . This button will turn off the marker alignment. 
• Select the Comparison context and create a comparison file of a sample or ladder where you suspect late migration and the ladder from the demo file.

With the analysis off, we would not anticipate the markers overlaying perfectly. However, clear delay in migration as shown in Figure 2 would be likely to cause problems with marker assignment. 

 
Figure 2. Comparison of a ladder affected by late migration (red) and a ladder from a demo file, which has migrated as expected (blue)

Late migration can lead to peaks not being detected automatically and if the peaks don't migrate within the assays time window for detection they will not be detected at all. If the upper marker and all ladder peaks were detected, just not assigned correctly, then you may be able to still use the data by reassigning the upper marker as described in this Agilent Knowledge Portal Article. If the upper marker or biggest ladder peak is not detected, then accurate sizing or quantitation of the sample peaks is not possible.

Late migration caused by: Problems during chip priming, dirty electrodes, liquid spillage from the chip, loss of gel separation properties or the dye concentration being too high.

Steps to Follow

To prevent the unnecessary consumption of multiple chips during troubleshooting, please read this article in full and perform all recommended maintenance before running another chip.

To exclude a problem with the chip priming

1. Check the priming station base plate and syringe clip settings for your assay, especially if the instrument is also used to run other assays.

2. Perform the maintenance of the priming station. Instructions are located in chapter 9 of the Maintenance and Troubleshooting guide and in the Bioanalyzer how to video: priming station maintenance (Figure 3).

   • Exchange the silicone gasket if it hasn’t been changed for three months or if it shows any signs of damage or clogging. The part number of the new gasket kit is G2938-68716. 

   • The syringe should be changed quarterly or whenever it is clogged, and its part number is G2938-68706.

   • Visually inspect the priming station's syringe adapter for any indication of a blockage. 

     
     
     Figure 3. Bioanalyzer how to video: priming station maintenance
     
     

3. Prime and check the underside of the chip for any bubbles in the channels. Do not turn the primed chip upside down, rather check it by holding it up. In a properly primed chip, the microchannels will not be visible, the presence of bubbles would indicate a priming problem.

To exclude a dirty electrode cartridge

• Perform the full maintenance of the electrode cartridge as outlined in chapter 8 of the Maintenance and Troubleshooting guide and in the Bioanalyzer how to video: electrode cartridge maintenance (Figure 4).
• Dry the pin set after cleaning with compressed air or leave the pin set in a desiccator overnight.
  • If the instrument is used regularly, Agilent recommends cleaning the pin set thoroughly every month.
  • Clean the pin set after any prolonged period during which the instrument was not in use.

Caution: Make sure that the pin set is fully dry before placing it back into the electrode base. Even small amounts of liquid on the pin set can damage the high-voltage power supply. Before starting a run, run the short circuit test as described in chapter 4 of the Maintenance and Troubleshooting guide.

 

Figure 4. Bioanalyzer how to video: electrode cartridge maintenance

To exclude a problem with the chip preparation

• Check if the gel and gel-dye mix could be expired or could have been stored incorrectly. Loss of gel separation properties can cause late migration. Make a fresh gel-dye mix before running the next chip to be on a safe side.
• Make sure that the reagents are warmed up for 30 minutes to between 20-25^oC before preparing the chip. Store chips at room temperature. The reagents have been validated at room temperature and a lower temperature will affect the separation properties of the gel.
• Check the surface of the chip for any liquid spills after the vortexing.
  • If liquid has been spilled, reduce the vortexing speed to 2000 rpm.
  • Chips should be vortexed for one minute using the IKA vortexer.
  • Check if there are any detergents in your sample buffer, which could reduce the surface tension of the liquid in the well. If detergents are present, vortex 5 μl of buffer and 1 μl of sample in a vial. Then spin down the vial and load this 6 μl onto the chip. 

To exclude a problem with the samples

• After you have considered the advice in this article and performed the priming station and electrode cartridge maintenance, prepare a chip without samples. As this chip doesn't contain any samples, they will not influence the migration of the markers and ladder. 
• For RNA and DNA assays 6 μl of marker should be pipetted into each sample well. 5 μl of marker + 1 μl ladder should be pipetted into the ladder well.
• If the late migration persists, send the affected data files to your Agilent support representative. The Bioanalyzer data files have a .xad extension and are by default saved in the following location: C:\Program Files (x86) \Agilent\2100 bioanalyzer\2100 expert\Data.

Learn how to effectively maintain your Bioanalyzer: Agilent 2100 Bioanalyzer System Maintenance and Troubleshooting Guide on Agilent.com

For Research Use Only. Not for use in diagnostic procedures.

PR7001-0968