DNA & eDNA

Equipment and Materials

FIELD COLLECTION FILTRATION FILTER PRESERVATION
Polypropylene (PP or HDPE) Nalgene sample bottles, 1 L size 250 mL filter funnels with 47 mm diameter, 0.45 µm pore size cellulose nitrate membrane Paper coin envelopes (3”x5”) and silica bead desiccant or 2 mL cryogenic vials with 0-ring and 95-100% molecular grade ethanol
Permanent marker High-capacity vacuum pump (e.g. Gast vacuum pump with 1.1 cfm, 25.5” Hg, 115 VAC) Ziplock bags
Non-powdered, disposable gloves (nitrile or latex) Appropriately sized silicon tubing to connect vacuum flask to the vacuum pump Permanent marker
Tote with a 10% bleach-water solution 1 L vacuum flask  
Scrub brush #8 rubber stopper  
Notebook 2 pairs of stainless steel, straight forceps/tweezers  
GPS unit 50% bleach solution  
Cooler with ice or reusable ice packs Deionized/distilled water  
Optional: 14’ extendable pole with bike water bottle cage attached 3 rinse containers to wash forceps  
  1 L measuring cup  

For specific equipment and materials suggestions contact us at 1.877.706.7678 / 519.836.2400 ext. 7067714.

Collection Procedure

  1. Using a permanent marker, label all sample bottles with the relevant information (ie: site ID, sample ID, etc). Be aware that more than one sample bottle may be required, depending on the field sampling design.
  2. Put on a new, clean pair of gloves at every collection site and avoid having contact between the gloves and anything other than the collection bottles.
  3. Rinse all of the sample bottles and lids three times each by collecting surface water close to the sample collection location, lightly closing the lids and shaking the bottles. Discard this water away from the sampling location (downstream or on the shore/ground).
  4. Fill the sample bottles with 1 L of water from the surface of the collection site and tighten the lids. Immediately place these bottles in the cooler on ice.

Filtration Procedure

  1. Run the tubing from the pump to the vacuum flask, attach the rubber stopper to the vacuum flask and plug the pump into the power source.
  2. Insert the plastic funnel adapter into the rubber stopper so that the seal is airtight. Remove the funnel filter from its packaging and attach it to the adapter. Be careful not to touch the inside of the funnel as you may contaminate it.
  3. Gently invert the sample collection bottle a few times to mix the sample, then slowly pour the sample into the filter funnel until the funnel is filled. Take note of the volume dispensed and filter start time.
  4. Turn the pump on to begin filtration. Add sample water in small increments as the vacuum draws water into the flask during filtration to avoid having to pour out any unfiltered water that may not pass through if the filter becomes clogged and filtration rate slows or stops.
  5. When filtering is complete, use the measuring cup to record the volume of sample water that passed through the membrane into the flask and record total filtration time.

Preservation Procedure

  1. Using a permanent marker, label a coin envelope (for dry silica storage) or a 2 mL tube/vial of ethanol (for ethanol storage) with the appropriate sample information (site ID, sample ID, etc.)
  2. Prior to removing each sample filter from the filter funnel, clean two pairs of forceps in a cup containing 50% bleach solution for at least one minute then swirl them in a different cup of deionized/distilled water. Next, place them in a second cup of deionized/distilled water for at least 30 seconds. This should prevent both contamination and damage to eDNA from residual bleach. It is very important that all traces of bleach be removed.
  3. If preserving your sample in silica, be sure that the membrane is dry by pulling air through the filter for 3-5 minutes with the vacuum pump after all water has passed through. If preserving in ethanol, the membrane need not be completely dry.
  4. Gently break the seal between the filter funnel and the funnel adapter by pulling the filter funnel upwards – do not twist the filter funnel as this could damage the sample membrane and result in sample loss.
  5. Using the clean forceps, carefully fold the filter membrane in half once (for silica preservation) or twice (for ethanol preservation) with the filtrand on the inside of the fold.
  6. Place the folded filter into the coin envelope or ethanol-filled vial/tube. Place the coin envelope into a Ziplock bag with silica beads and seal the bag. Place the ethanol vial/tube into a Ziplock bag and seal the bag. Write the appropriate information on each Ziplock bag. To help prevent contamination, keep coin envelopes from different sites in different Ziplock bags.

References

Jared Hobbs and Caren Goldberg, Environmental DNA Protocol for Freshwater Aquatic Ecosystems, Version 2.2. BC Ministry of Environment, Ecosystems Branch Victoria, BC. November 2017.

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