No Data From One or More Capillary on Parallel Capillary Electrophoresis Instruments

Answer

This Information Applies To: 5200, 5300 and 5400 Fragment Analyzer, Femto Pulse and ZAG DNA analyzer

Issue

When no data is available in the Agilent ProSize data analysis software for one or more capillaries, the capillary(ies) could be misaligned, blocked, require extra conditioning, or the electrodes might be encrusted.

Steps to Follow

Check the capillary alignment

Check the alignment of the capillaries in ProSize. Checking the capillary alignment is briefly described in this section, but is described in full in Chapter 9 of the ProSize Data Analysis Software User Manual.

1.  Click Analysis>View Capillary Positions.
2. In the View Capillary Positions window, if the alignment is correct, the red dots will be at the top of each blue peak.
3. If the alignment is not correct but a blue peak is present for each capillary array (e.g there will be 12 peaks for a 12 capillary array), drag and drop the red dots onto the top of the blue peaks.
4. If there are fewer blue peaks than capillaries in the capillary array, review the subsequent section. 
5. Once you are happy with the capillary alignment, click Accept to confirm the current capillary positions and return to the main ProSize menu. This will correct the alignment for this data file only.

If you have corrected the capillary alignment and you now have data for the missing capillary or capillaries, you have identified the root cause of the problem. Correct the alignment using the instrument controller software to avoid the alignment being incorrect in future data files. Check chapter 4 of the Fragment Analyzer System Manual, Femto Pulse User Manual, or ZAG DNA Analyzer System Manual for instructions. It is recommended and easiest to follow the Capillary Alignment from a File method. The capillary alignment should be done with the new data file, as only this latest run will have the current capillary positions. 

If the signal from one or more capillary is missing

First, check for the presence of a blockage. A blockage can be confirmed by running a full conditioning method with a deep-well plate in the waste drawer. The conditioning method requires gel and Conditioning Solution. To start the conditioning method, from the operation tab in the controller software, click Add to queue under the heading Capillary Array - Conditioning. The Select Conditioning Method window will open and the Full conditioning.mthdc can be selected from the drop-down menu (Figure 1). If a capillary is blocked, no waste, or less waste compared to the functioning capillaries, will be present in its corresponding well.

Figure 1. Running a full conditioning method in the controller software.

If a capillary is blocked

Caution: To prevent blocked capillaries, the capillary array should be stored in Capillary Storage Solution. The Capillary Storage Solution should be changed at least once every four weeks. However, it is recommended to change the Capillary Storage Solution every one to two weeks due to evaporation, especially in low humidity/high temperature environments. It is best practice to run a full conditioning run weekly.

To unblock the affected capillaries, perform a Method B - hot water soak followed by a Method C - 0.5N NaOH flush. How to perform these methods is described in the appendix of the 5200, 5300, and 5400 Fragment Analyzer, Femto Pulse, and ZAG DNA Analyzer system manuals.

 During the Method C flush, the NaOH concentration can be increased to 1 N but no further. Flushing the array repeatedly with 1 N NaOH may damage the capillaries and should therefore be avoided if possible. If the Method B and Method C flush do not unblock the affected capillaries, a new capillary array may need to be purchased.

If no capillaries are blocked

The electrode of the affected capillary could be encrusted with, for example, dried Storage Solution, or the capillary might require extra conditioning.

1. To remove any encrusted solution, perform a Method B - hot water soak followed by a Method C - 0.5N NaOH flush. These methods are described in the appendix of the 5200, 5300, and 5400 Fragment Analyzer, Femto Pulse, and ZAG DNA Analyzer system manuals.
2. Before the next run, run a full conditioning method. 

If the Method B and Method C flush and extra conditioning does not restore the signal of affected capillaries, a new capillary array may need to be purchased.

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

PR7001-0840

Learn how to effectively maintain and operate your parallel capillary electrophoresis instrument: 5200, 5300, and 5400 Fragment Analyzer System Manual on Agilent.com Femto Pulse User Manual on Agilent.com ZAG DNA Analyzer System Manual on Agilent.com ProSize Data Analysis Software User Manual on Agilent.com