Struggling with Sterivex and finding small solutions that make lab life easier (by Kari-Anne van der Zon)

by Kari-Anne van der Zon, PhD candidate at Strasbourg University in Strasbourg, France and 2023 eDNA Collaborative Microgrant recipient

I started my PhD in this strange just-after-covid period where plastic labware seemed scarce. It was April and my supervisors wanted me to sample aquatic eDNA in June. We did not really know what materials would be best so I hurriedly read some papers and asked a friendly scientist for advice. Festina lente. In the back of my mind a voice was telling me that it is not a good idea to make decisions quickly. But I needed to take samples. Lost in the aquatic eDNA literature I was relieved to stumble upon Spens et al. (2017) and decided that Sterivex filter capsules preserved in Longmire’s buffer would do the job.

Given the plasticware-scarce situation we felt relieved the filters arrived some weeks before we boarded the airplane in to sample Latvian ponds in June. I was quite happy with the suitcase containing Sterivex units that would be handy in the field and Longmire’s buffer that would preserve the DNA until we could freeze the filters back in the lab. I also had packed UV-cleaned parafilm and plastic bags. These were needed to wrap the filters in after sampling because I had not managed to find outlet caps for the filters I had bought. As I had had no idea what Luer-lock meant when ordering the filters I had bought Sterivex filters with normal Luer, not Luer-lock outlets, and caps fitting these outlets seem impossible to find.

The wrapping in parafilm was a bit of a hassle in the field but not a great problem. I found that I could neatly store my filters after capping and wrapping with the capped inwards down in cryoboxes and was content. The trouble started when it was time to extract DNA. It was nice to have a plan for steps A, B and C, but steps D to Z appeared to exist as well. My supervisor had given me an extraction protocol. For normal filters. Filters you could put in Eppendorf tubes. Not for these hard plastic units I had filled the freezer with.

A PhD colleague suggested opening the Sterivex capsule. We even found a paper by Cruaud et al. (2017) about opening the Sterivex filter capsule. We cleaned some cutters thoroughly with DNA-exitus and went for it. It was a mess. Splashes of sample scattered around the UV hood. Cruaud et al. (2017) opened their capsules with an autoclaved PVC tube cutter but the logistics of this seemed unfit for our large number of samples. The DNeasy PowerWater Sterivex Kit was quite out of our budget. In the end we decided to just try to perform the first extraction steps in the filter capsule. Luckily Sterivex units do fit in everything that is made for 15 mL Falcon tubes. Luckily we already had lysis buffer in the capsule so we didn’t have to pipette soapy buffer into the units.

In the beginning I closed the outlet caps by melting them over a flame. This works but is cumbersome. Our project manager heard me complain about the impossibility to find fitting outlet caps and pointed out that you can place a piece of catheter tube inside a non-fitting Luer-lock outlet cap and make it fit. Now our lab also has a second 15 mL Falcon ThermoMixer block so I can perform lysis of two batches at the same time. Also I learned that 2 mL syringes are very handy for getting lysate out of the Sterivex capsule. The extraction process still goes slower than I expected and if I would start another eDNA project I would sit down with a cup of tea and ponder over the whole process before I would order Sterivex again. However, with the small solutions the extractions do go smoothly now and I am looking forward to the next metabarcoding steps.

Figure 1: Capping a Sterivex outlet by placing a piece of catheter tube in a non-fitting outlet cap

Figure 2: Using 2 mL syringes to get liquid out of the Sterivex after lysis in the ThermoMixer

References

Spens, J., Evans, A. R., Halfmaerten, D., Knudsen, S. W., Sengupta, M. E., Mak, S. S., ... & Hellström, M. (2017). Comparison of capture and storage methods for aqueous macrobial eDNA using an optimized extraction protocol: advantage of enclosed filter. Methods in Ecology and Evolution, 8(5), 635-645.

Cruaud, P., Vigneron, A., Fradette, M. S., Charette, S. J., Rodriguez, M. J., Dorea, C. C., & Culley, A. I. (2017). Open the SterivexTM casing: An easy and effective way to improve DNA extraction yields. Limnology and Oceanography: Methods, 15(12), 1015-1020