Wavy Baseline on the Agilent Bioanalyzer

Answer

This Information Applies To: 2100 Bioanalyzer, 2100 Expert software

Issue

The Agilent 2100 Bioanalyzer system electropherograms in the Agilent 2100 Expert software have a wavy, uneven baseline (Figure 1).

 

Figure 1. Wavy, uneven baseline on Bioanalyzer electropherograms

The error is most often associated with:

• Problems during chip priming
• Dirty electrodes
• Moisture on the electrodes
• Sample contamination
• The sample concentration being higher than the assay's range
• Liquid spillage from the chip wells

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

• Check the priming station base plate and syringe clip settings for your assay, especially if the instrument is also used to run other assays.
• Perform the maintenance of the priming station, following the instructions in chapter 9 of the Maintenance and Troubleshooting guide and in the Bioanalyzer how to video: priming station maintenance (Figure 2). Exchange the silicone adapter gasket if it hasn’t been changed for three months or if it shows any signs of damage or clogging. If you do not have any gaskets left, you can order a new gasket kit from Agilent, the part number is G2938-68716. The syringe should be changed quarterly or whenever it is clogged. A new syringe is provided with each purchased Bioanalyzer assay kit and its part number is G2938-68706. The required frequency of the priming station maintenance procedures is described in this article.
• Visually inspect the syringe adapter of the priming station for any indication of a blockage. The narrow channel can get blocked if gel material is accidentally pulled inside after the chip priming. If there is only a small amount of material, it could cause a temporary blockage and intermittent problems. 
• After priming, 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 compared to the channels on a chip that has not been primed. The presence of bubbles would indicate a priming problem.

Figure 2. Bioanalyzer how to video: priming station maintenance

To exclude a dirty or wet 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 3). Make sure that the pins are completely dry after cleaning (dry them with compressed air or leave 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.
• Use only 350 μL of water in the cleaning chip.
• Ensure that the humidity in the room is below 70% at 15–27 °C (59–81 °F).

Caution: Damp pin set: 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, check that the pin set is dry by running the short circuit test as described in chapter 4 of the Maintenance and Troubleshooting guide.

Figure 3. Bioanalyzer how to video: electrode cartridge maintenance

To exclude a problem with the chip preparation

• Make sure that the reagents are warmed up for 30 minutes to 20–25 °C before preparing the chip. Store chips at room temperature.
• Check that the gel and gel-dye mix aren't expired or stored incorrectly. Make a fresh gel-dye mix before running the next chip and use dye concentration according to the assay quick start guide.
• Check the surface of the chip for any liquid spills after vortexing and verify that the vortexer is shaking in only one plane. If liquid has been spilled, reduce the vortexing speed to 2000 rpm. Check if there are any detergents in your sample buffer, which could reduce the surface tension of the liquid in the well.

To exclude a problem with the samples

The wavy baseline symptom can be sample-related. Chip overloading and contamination with high molecular weight molecules can cause a wavy baseline.

• To avoid overloading the chip, the samples must be within the assay's specified concentration range. Check the concentration of your samples by another method.
• Check the DNA or RNA isolation protocol to exclude contamination with high molecular weight DNA or protein. To remove genomic DNA from RNA samples, perform a DNase treatment.
• After you have performed the priming station and electrode cartridge maintenance, prepare a chip without samples to check if they are contributing to the wavy baseline. 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.

To exclude a problem with the instrument

• Changes in ambient temperature of more than 5 °C during the run can cause wavy baseline. Place the 2100 Bioanalyzer instrument in a thermally stable environment.
• Rarely, an instrument defect can cause the wavy baseline symptom. If the wavy baseline persists after considering all the previous bullet points and conducting the recommended maintenance, run the complete list of hardware diagnostic tests. The hardware diagnostics require an in-date set of test chips, part number G2938-68300. How to run the hardware diagnostics and send the results to Agilent is described in this article. If any hardware tests fail, and/or the wavy baseline persists send the hardware diagnostic results and the affected data files to your Agilent support representative. The Bioanalyzer data files have an .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

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

PR7000-8834