Troubleshooting unable to measure flow rate errors on the MiSeq

If the MiSeq fails to measure flow rate when starting a run, perform the following steps to resolve the issue. Steps 1 and 2 can be performed together, before repeating the flow check.

Troubleshooting steps:1. Inspect the flow cell/flow gaskets and the manifold for any signs of damage: 1. Open the MiSeq door and remove the flow cell. 2. Inspect the manifold for any visible cracks. * The flow cell manifold is an amber/yellow colored piece of plastic above the flow cell stage. If any cracks are suspected, email Illumina Technical Support with a picture of the manifold. 3. Inspect the flow cell to confirm that the flow cell gasket is flush, as shown in the following image (left). *

4. If the gasket is raised or distorted, do not use this flow cell. Email [Illumina Technical Support](mailto:[e-mail]) to receive a replacement for the defective flow cell.    
  * Technical Support will ask for the lot and serial numbers of the affected flow cell and may ask for a picture of the defective gasket for quality control.  
  * Proceed with the pre-run checks using a new flow cell from another kit, if available.  
5. If the gasket is flush with the plastic cartridge, clean and reseat the flow cell.  

2. To clean and re-seat the flow cell: 1. Lightly rinse the flow cell with laboratory-grade water until both the glass and plastic cartridge are thoroughly rinsed of excess salts. 2. Using care around the black flow cell port gasket, thoroughly dry the flow cell and cartridge with a lint-free lens cleaning tissue. Gently pat dry in the area of the gasket and adjacent glass. 3. Clean the flow cell glass with an alcohol wipe, but do not use alcohol on the gasket. 4. Check the flow cell stage for any debris or salt and clean the flow cell stage using an alcohol wipe or a lint-free tissue moistened with ethanol. 5. Return the flow cell to the stage and ensure it is seated properly. 6. Using MiSeq Control Software, perform another flow check. 3. If the flow check still fails, remove any reagents and power cycle the instrument. 1. Shut down Windows using the MiSeq Control Software Menu and turn off the instrument using the power switch on the back panel. 2. Leave the instrument off for at least 2 minutes to allow all parts to power down completely. 3. Turn the instrument to ON using the power switch on the back panel. 4. Using MiSeq Control Software, perform another flow check. 4. If the flow check still fails, use a different flow cell (from another kit). 1. Open the MiSeq door and remove the flow cell. 2. Clean the flow cell as described in step 2. 3. Place the new flow cell on the stage and ensure it is seated properly. 4. Using MiSeq Control Software, navigate back in the run set up to repeat the flow cell RFID check. This will record the new consumable information. 5. Continue with the run setup and perform another flow check. 6. If the flow check passes, record the lot number and serial number of the flow cell that didn`t work and email Illumina Technical Support to receive a replacement for the defective flow cell. 5. If the flow check still failing, an engineer service visit is needed and instrument fluidics is suspected. To test fluidics and collect information for the engineer, complete a manual post-run wash with a previously used flow cell, complete a system check, and email Illumina Technical Support with the results for further assistance. 1. **The new/unused flow cell should not be used to complete a wash on the instrument. Remove the flow cell from the instrument, return it to the buffer, and place it at 4°C in the refrigerator. 2. Use a previously used flow cell to complete the post-run wash and system checks on the instrument. 3. Follow the guidelines on Reagent Stability and Thawing Guidelines for MiSeq Sequencing Kits to salvage consumables when possible. 6. If the issue persists with a new flow cell, and after completing a wash, and system check, email Illumina Technical Support for further assistance.

For further information, search for Knowledge Base articles: How to shut down or power cycle the MiSeq Wash best practices for the MiSeq How to perform a System Check on the MiSeq

For any feedback or questions regarding this article (Illumina Knowledge Article #1431), contact Illumina Technical Support [e-mail].