Troubleshooting Flowcells

Contents

1. Overview
2. Steps to try
3. Notes

1. Overview

Periodically, your flowcells will stop working. Frequently, this occurs after taking a prolonged break from the microscope. Some symptoms may include: a. DNA stuck everywhere, b. High QD background, or c. very few DNA molecules immobilized on the surface. In our old lab (Green Lab), we used to call this “flowcell rot” disease.

The precise cause of “flowcell rot” is still unknown. However, these simple steps typically got me back on track:

2. Steps to try

1. Re-make your buffer stocks. Keep small amounts dedicated for microscope-use only.
   1. 1M Tris pH 8 (filter-sterilize a 0.2-0.5L stock in a SteriCup. pH should not be below 8)
   2. 1M MgCl2 (filter-sterilize 0.1L stock)
   3. 5M NaCl (filter-sterilize 0.5L stock)
2. Re-make lipids, if possible from fresh DOPC/mPEG stocks.
3. Use fresh scratched quartz slides to check DNA quality and lipid fluidity.
   1. If you see good DNA curtains, this suggests you have good buffers/lipids.
4. Re-wash your nano-fabricated slides. Putting the flowcells in 100% EtOH overnight can help to improve the fluidity.

3. Notes

You can also try re-making fresh BSA, Streptavidin, and Gloxy. However, I’ve always felt that fresh buffers and lipids make all the difference.

Yoori‘s note: In my experiences, washing the flowcells thoroughly by taking a few days often resolved the ’flowcell rot’ issue and improved the quality of DNA curtains. (washing with Hellmanex overnight, storing in oven overnight, storing in EtOH overnight before use.)

With practice, your flowcell success rate should exceed ~80-90%.