SAMPLING PROTOCOL

GREAT CRESTED NEWT eDNA KIT

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USING THE KIT

KIT CONTENTS

Kit contents are based on one person collecting an eDNA sample. If two or more people will be handling kit contents and collecting an eDNA sample, please contact us about inclusion of extra gloves in kits. Extra gloves are not provided as standard to minimise waste.

2x pairs of nitrile gloves

1 x 1.6 L sampling bag
1 x resealable bag

1 x 30 mL ladle dipper

1 x plastic pipette

1 x sampling datasheet

6 x 50 mL tubes, each containing 35 mL of ethanol and preservative, inside cardboard box with insert

PREPARATION

Download and log into the NatureMetrics app over Wi-Fi before arriving in the field.

IN THE FIELD

Gloves should be worn at all times during the sampling process.

Samples should be collected from the shoreline without entering the water. This prevents disturbance of the substrate and limits cross-contamination.

USER PROTOCOL

1. Identify where you will take 20 subsamples from the pond. Subsamples should be spread out evenly around the pond perimeter. If some of the pond perimeter is inaccessible, subsamples should be spread out evenly around accessible areas. Both open water (displaying habitat) and vegetated (egg-laying substrate) areas (if present) should be represented by the subsamples.

2. Put on one pair of provided gloves. Open the provided sampling bag by tearing off the clear plastic strip along the perforated line, then pulling the tabs to open the bag.

3. Collect 20 x 30 mL subsamples of pond water from around the pond (see step 1) using the provided ladle (fill the ladle to the top) and empty each subsample into the sampling bag. At the end, the sampling bag should be approximately half full (600 mL).

Before each ladle of water is taken, the pond water column should be gently mixed. Use the ladle to stir the water from the surface to close to the pond bottom without disturbing the sediment on the pond bottom. It is advisable not to sample very shallow water (less than 5-10 cm deep). The sampling bag is not self-standing so it should be propped against a rock/log/tree stump when it needs to be set down.

4. Once 20 subsamples have been taken, roll down the top of the sampling bag to seal, and shake the bag for 10 seconds. This mixes any DNA present in each subsample across the entire water sample.

5. Put on a new pair of gloves to minimise contamination risk to the next stage.

6. Before taking each aliquot, swirl the water in the sampling bag to homogenise the sample – this is because DNA will constantly sink to the bottom. Using the clear plastic pipette provided, take a 15 mL aliquot of water from the sampling bag and pipette into a sterile 50 mL conical tube containing 35 mL of ethanol and preservative (i.e. fill tube to the 50 mL mark). Close the tube, ensuring the cap is on tight. Shake the tube vigorously for 10 seconds to mix the water with the ethanol and preservative, then place it back into the cardboard insert.

7. Repeat step 6 for each of the 6 tubes in the kit. Seal the cardboard box containing the samples.

8. Scan the barcode on the box using the NatureMetrics app and submit the data for your samples. Complete and sign the sampling datasheet. Please record your sampling data on paper and through the app so there is a physical and electronic copy of your data.

9. Place the cardboard box with samples and the sampling datasheet inside the resealable bag. Empty the remaining water in the sampling bag back into the pond. Dispose of all waste responsibly.

10. The samples are collected and couriered to the NatureMetrics laboratory at ambient temperature. If samples are stored overnight before shipping, they should be refrigerated at 2-4°C. Samples can be stored for up to one month in the refrigerator before analysis, but should not be frozen.

Please ensure you have booked your sample collection and analysis on my.naturemetrics.co.uk.

Contact our GCN team for support at gcn@naturemetrics.co.uk.

PACKAGING NOTES AND QUICK LINKS

  • Return your samples using the box that kits were sent in, where possible.

  • If using an alternative box, ensure that it is in good condition and remove any old labels.

  • Avoid excess spacing by adding cushioning material (e.g. bubble wrap, newspaper) to avoid the product shifting inside the box.

  • Use good quality sealing tape and seal along box edges in a H pattern.

  • The only essential thing to send back to us is the resealable bag containing the cardboard box with samples and the sampling datasheet. However, all kit contents can be sent back to NatureMetrics for recycling.

  • Register for your my.naturemetrics account here.

  • Please find our Terms and Conditions here.

CLIENT RESPONSIBILITIES

Important considerations for your sampling

  • Samples from some locations might have compounds that inhibit analyses resulting in reduced data generation – while we routinely test for inhibition, it is not always possible to overcome.

  • It is the responsibility of the client to ensure that all efforts have been made to avoid contamination from external sources and between samples.

  • Handling samples without gloves can increase the content of human DNA, reducing data generation.

  • After sampling, movement of samples across borders without permission is not allowed as they are classed as biological samples.

Disclaimer: Safe sample collection is the responsibility of the Client. NatureMetrics accepts no liability associated
with the use of the kits and sample collection. The Client is solely responsible for the quality of the samples and the representativeness of the samples received by NatureMetrics. Please read the full limitation of liability statement in the Terms and Conditions.

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