Performing Capillary Alignment Using Run Data on Agilent Parallel Capillary Electrophoresis Instruments

Answer

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

Issue

Perform a capillary alignment whenever a new array has been installed on an Agilent Parallel Capillary Electrophoresis instrument. An alignment is also needed if the capillary positions suggest that the signal is not captured at the highest intensity for all the capillaries. 

Steps to Follow

1. Open the Instrument controller software and in the Utilities drop-down menu, select Capillary Alignment. (Figure 1)

   
   
   Figure 1. Select Capillary Alignment 
   
   

2. Select No when prompted with the option to fill the capillary with dye from the Capillary Alignment Prep window. (Figure 2)

   
   
   Figure 2. Capillary Alignment Prep window
   
   

3. From the top menu bar of the Capillary Alignment pop-up window, select Read Raw.
4. Select the raw data file that you wish to use for the alignment using the Windows file explorer. (The default-saved location of raw data is C:/Agilent/Data/(Date: YYYY MM DD)/(Time: HH MM).
5. Select the latest Raw file (e.g. the test plate/test row).
6. From the align-from-file pop-up window, left click the red baseline and drag it upwards off the bottom of the graph, but not above the top of any of the capillary peaks. (Figure 3)

   
   
   Figure 3. Align from file pop-up window for a 12-capillary system 
   1. Capillary pixel locations. 2. Yellow boxes denote capillary location, 3. Baseline
   
   

7. Select Locate Caps from the Align from File Toolbar (Table 1). The capillary peaks will be located and a yellow box is placed at the apex of the selected capillaries denoting the capillary pixel location.         
                    

   Icon	Description
   Open	Opens a new file.
   Ok	Accepts changes to the file (i.e., capillary locations).
   Cancel	Cancels any actions and closes the file.
   Original	Locates the original capillary positions used when the selected file ran.
   Locate caps	Locates the capillaries based on peak positions in the selected open file. Note: Move the red baseline so that only the peaks of interest are integrated and not noise from the baseline.

   Table 1. Align from file toolbar functions
   
   

8. The number of capillaries found should be 12, 48, or 96 depending on the configuration of the instrument and the type of array in use.                                                                                                                         
9. If necessary, adjust the capillary positions:
   • To manually adjust a capillary position, left-click on the white line showing the capillary position and drag it left or right to the desired location.
   • To zoom-in for the desired resolution, right-click, select Zoom, and select the appropriate area.
   • Should the number of detected capillaries be incorrect, adjust the red baseline, and repeat the preceding steps.
   • To insert or delete a capillary position, right-click the black area of the graph or the capillary pixel location table to the right of the graph.
10. Select OK from the Align from File Toolbar once the desired number of capillaries are located.
11. Select Save from the main Capillary Alignment Screen. From this point forward, the instrument will use these saved pixel locations for all future runs. 

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 Oligo Pro II User Manual on Agilent.com ZAG DNA Analyzer System Manual on Agilent.com

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

PR7001-1613