HOW TO AVOID INCONCLUSIVE RESULTS

Advice Note

Occasionally results will be reported as inconclusive. Inconclusive results may arise because target species’ DNA was not detected, DNA was degraded, or samples were inhibited.

Please read our guidance below for how to avoid DNA degradation and prevent inhibition.

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Kit Contents

Vampire Pump

This can be hired from NatureMetrics.

1 x pump head (A)

1 x drive unit (B)

Degradation

DNA degradation is a process by which DNA has been damaged to the point that it cannot be detected. The use of preservatives in our kits minimizes the risk of DNA damage, however, inappropriate storage conditions may still lead to DNA degradation. Below are guidelines to minimize DNA degradation after sampling. Degradation of DNA after sampling is unlikely if the guidelines below are followed.

Minimizing the risk of degradation in samples:

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Degradation factor	Details	Tips to minimize risk of degradation
Time	DNA naturally degrades over time – please return your samples to us as quickly as possible.	For aquatic kits, ensure that all water has been expelled from the filter, all the preservative is pushed into the filter, and filters are frozen if they cannot be returned to us within 2 weeks of storage at ambient temperature. For Soil/Sediment Kits, use cold storage transportation for samples (preservation buffer kit available if cold storage is not feasible) and return them to us within 24 hours via the fastest service available (next day delivery where possible).
Heat	Warm temperatures can speed up DNA degradation.	Filter water immediately after water samples are collected and keep filters cool. Increase the number of sampling replicates if sampling in warmer seasons or climates.
UV exposure	UV light is capable of directly degrading DNA or reacting with organic matter to cause oxidative damage to DNA.	Store samples out of direct sunlight after collection. Increase the number of sampling replicates if sampling in areas of high UV intensity.
Water chemistry	Conditions that are favorable for microbial growth, such as neutral pH and higher dissolved oxygen, are more likely to promote DNA degradation.	Increase sample replication to ensure that you are collecting as much DNA as possible in your samples. If possible and appropriate, measure water chemistry parameters after eDNA samples have been collected and record values on the sampling datasheet to help with interpretation of results.

Inhibition

PCR inhibition occurs when compounds present in the sample (e.g. acids) interfere with the PCR reaction, preventing or reducing target DNA amplification and subsequent detection. Inhibitors may interact directly with target DNA or interfere with the enzyme that drives the PCR reaction. We highlight ways to minimize the occurrence of inhibition in the table below. 

Minimizing the risk of inhibition in samples:

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Known inhibitor	Details	Tips to minimize risk of inhibition
Humic/fulvic acids	Organic compounds found in soil, peat, and coal.	Avoid stirring up sediment.
Organic debris	Animal waste.	Avoid sampling where there are obvious signs of livestock nearby.
Tannins	Compounds found in wood, bark, leaves, and fruit of plants.	Avoid capturing plant debris, leaf litter or duckweed in samples.
Calcium carbonate	Abundant in rocks such as chalk and limestone.	Avoid waterbodies with high chalk content (i.e., milky/cloudy water).
Chemical pollutants	Can come from industrial work, agricultural runoff etc.	Avoid oil slicks and avoid sampling when agricultural sprays are in use nearby.
Sediment	Material at the bottom of a waterbody which can clog filters, reducing DNA collection, and contains humic/fulvic acids.	Avoid stirring up sediment, and avoid areas of sediment accumulation (e.g., in high-flow rivers).