Collection and preservation of eDNA from marine water samples
Ana Ramón-Laca, Abigail Wells, Linda Park
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Abstract
This protocol is designed for water collection from Niskin bottles and filtration at sea using reusable filter cups.
Aim: to collect and filter 2.5 L of water at each depth from each CTD cast and preserved the filter at room temperature in lysis buffer
Steps
Filter cups cleaning and assemblage
Set up wash station (two buckets, one with 0.5% bleach, one with clean distilled H2O)
Place all used small items in a small mesh bag and the filter cups in a large mesh bag in bleach for at least 0h 30m 0s
Rinse the abovementioned items for at least 0h 10m 0s
Allow to dry on the drying racks
Wearing a fresh pair of gloves, mount filter cups with new sterile filter
Collection of water from Niskin bottles
Let the water from the hull (“surface water sample”) run for > 0h 3m 0s while the CTD is being deployed
Collect 2 samples of2.5Leach of “surface water” for each CTD cast in 69oz whirlpack bags. Use pitchers for support and stability.
Once the CTD rosette has been brought back to the surface, water needs to be extracted from each Niskin bottle and transported into the lab for filtration.
We want to collect 2.5Lof water from each Niskin.
Wipe the spigot on the Niskin with DNAway to remove potential contaminants on the outside of the Niskin
Flush water (count to 10) from the Niskin to further reduce risk of contamination and collect 2.5LL of water in a whirlpack bag and place in 2L pitcher
Include a sampling negative control by collecting 2.5L of distilled H2O (at least daily)
Filtration of water samples
Wipe working area with DNAway. Use a clean absorbant pad each day for the filtration station.
Switch on the pump and set vacuum in the middle range between-8 and -12 bars (keep an eye on the gauge & adjust if necessary)
Pour water from whirlpack bag (supported by pitcher) into the filter cup, repeat until finished (2.5L of water)
Using DNA-free forceps fold filter and place in pre-labeled tube pre-filled with preservative buffer (2mL, Longmire Buffer in a 5 ml LoBind tube).
Filter 2.5L of ship distilled H2O every time the cleaning distilled H2O is replaced as a sampling negative control
Every 2-3 days 2L of lab diH2O should be filtered as sampling negative controls
Note tube label in notes. Also note time filtered, membrane type, place filtered (field/lab, which lab), etc.
Store samples at room temperature away from UV light
