The overall goal of this protocol is to filter water samples with a filter cartridge and extract environmental DNA without having to cut open the housing to remove the filter from metabarcoding eDNA from various aquatic organisms. This method can help answer key questions in the ecological field, such as biodiversity monitoring and community ecology. The main advantage of this technique is that the filter cartridges can accommodate larger water volumes and the filtration and DNA extraction can be performed in a sterile manner.
To begin this procedure, attach high vacuum tubing to the input connector of an aspirator pump. Attach the other end of the tubing to a manifold. Be sure that the three red T-valves at the manifold are in the off position.
Next, put on a clean set of gloves. Insert a female luer fitting at the top end of the vacuum rubber tubing. Then, insert a vacuum connector at the bottom end.
Carefully attach the female luer fitting to an outlet port of the filter cartridge. Then, attach the vacuum connector to a silicone stopper. Pour one liter of sampled water into the prepared plastic bag.
Enclose the screw cap with the male luer-lock connector. Then, carefully connect an inlet port of the assembled filter cartridge with the male luer-lock connector. Ensure that all connections are secure.
Then, hang the plastic bag from two prongs on the mesh panel. Insert a silicone stopper into the inlet port of the manifold. Then, turn on the aspirator.
Open the red T-valves for filtration. Run the manifold until the filter cartridge is dry. Then, close the red T-valves.
Repeat this entire process to filter the desired amount of water. Once all of the sampled water has been filtered, carefully remove the filter cartridge from the plastic bag and vacuum container. Cap both ends of the cartridge with the appropriate luer caps.
Then, using a solvent proof pen, label the cartridge and inlet luer cap. Store at 20 degrees Celsius until ready to perform DNA extraction. For samples larger than four liters, prepare a ten liter book bottle as detailed in the text protocol.
Add the water sample to be filtered. Then, tightly insert a ten milliliter pipette tip into the valve of the book bottle. Tightly insert the inlet port of the filter cartridge into the pipette tip.
Then, insert a silicone stopper into the inlet port of the manifold. Turn on the aspirator. Then, open the red T-valves for filtration.
Run the manifold until the filter cartridge is dry. After the filtration is complete, close the red T-valves. Carefully remove the cartridge from the plastic bag and vacuum connector.
Then cap both ends of the cartridge with the appropriate luer caps. Use a solvent proof pen to label the cartridge and the inlet luer cap. Store at 20 degrees Celsius until ready to perform DNA extraction.
Preheat an incubator to 56 degrees Celsius. Then, cut the hinged cap from a 2.0 milliliter tube and discard the cap. Remove the inlet luer cap from the filter cartridge.
Then, insert the inlet port into the 2.0 milliliter tube. Using a self-sealing film, tightly seal the connection between the cartridge and the collection tube. Next, insert the combined unit into a centrifuge adapter for a 15 milliliter conical tube.
Centrifuge the cartridge for one minute at 5000 times g to remove the remaining liquid in the filter. Once centrifugation is complete, remove the collection tube from the filter cartridge and discard the tube. Then, recap the inlet port.
Next, mix 20 microliters of protein A-Case solution, 220 microliters of phosphate-buffered saline, and 200 microliters of buffer AL.Remove the inlet cap and add the mixture into the cartridge using a pipette, inserting the pipette completely into the inlet port. Recap the inlet port. Then, place the filter cartridge on a rotary shaker.
Place the rotary shaker in the preheated incubator, and set the shaker to 20 rpm for 20 minutes. During the incubation, label a new 2.0 milliliter tube with a solvent proof pen. Then, place it into a 50 milliliter conical tube.
After the incubation is complete, remove the inlet cap. Then, insert the inlet port into the new 2.0 milliliter tube. Cap the conical tube with a screw cap.
Centrifuge for one minute at 5000 times g to collect the extracted DNA. Then, remove the cartridge in the 2.0 milliliter tube using sterilized forceps. Cap the tube and purify the extracted DNA as outlined in the text protocol.
In this procedure, four different volumes of sea water are filtered on the same manifold using two unique filters, and eDNA is subsequently extracted. The filter cartridge detected, on average, 50%more species than the glass fiber filters. Once mastered, this technique can be done in two hours if it is performed properly.
After watching this video, you should have a good understanding of how to filter water samples with a filter cartridge, and how to extract eDNA without having to cut open the housing to remove the filter from metabarcoding eDNA from various aquatic organisms.
