RGG DNA extraction protocol for water samples (eDNA)

Research Group Genetics Authors: Kim Præbel (kim.pra[email protected]) Julie Bitz-Thorsen Norwegian College for Fishery Science Research Group for Genetics, K. Præbel Last updated: September 2023, edit KP EXTRACTION PROTOCOL FOR EDNA FROM STERIVEX FILTERS The protocol is using the Qiagen Blood and Tissue kit Guidelines and advises • Follow the descriptions in the ‘Clean Lab Routines’ of how to enter the labs. • Always start a project with a new extraction kit. The kit can only be used for that particular project and any remains are brought out of the lab and to the B310 lab when the extractions are completed. • Make sure that the incubator is set to 56oC before starting the extraction work. The equipment you are going to use for the extraction protocol should always be cleaned. • Make sure to check for any signs of crystals in the extraction buffers when opening the kit in the laminar flow-hood. If any signs, place the bottle(s) in the inclubator at 56oC until they are completely dissolved. • Always shake Eppendorf tubes out of the bag, don’t put your hand inside of it. Discard any excess tubes. • Only open the bags containing Eppendorf tubes, or other tubes, inside the laminar flow-hood. • Only use pipette tips with barriers/filters and only open the boxes inside the laminar flow-hood. • Always follow the workflow or any precautions given for the eDNA clean lab working routines. • Always discard tips/tubes/gloves if you have the slightest suspicion about contamination (e.g. if the tip touches the table before entering a tube or buffer bottle). • Always work with at least one extraction blanks per extraction round (i.e. 24 samples). However, if you are working with 22 samples to extract, then to complete the number to 24, you can work with two blanks. • Always start with the lowest concentration i.e. air blanks and water blanks (if any) except extraction blanks, which should be treated as any regular sample. • If extracting samples from several species/locations, sterilize everything between samples. • Do not touch the ends of the Sterivex filters or the inside of the tube caps with hands or tweezers. • Always be careful when you open the Eppendorf tubes not to touch the inside of the cap. Hold them in your hand and flick them open with the tip of your thumb. • MAKE SURE YOU HAVE ENOUGH TIPS ! - before entering the clean-lab. You will mainly use 1000µl tips but also stock up on 20µl and 200µl ones. You also need Eppendorf tubes (both 1.5ml and 2.0ml), 50ml falcon tubes. Always have enough of these things before you start working. DAY 0: 1. Follow the descriptions in the ‘Clean Lab Routines’ of how to enter the labs. 2. Since the dedicated eDNA -80oC freezer is located in a “contaminated” area, the preference is to take the samples out the day before. Then, the day after, you start your extractions by showing up in newly washed clothes and freshly showered. 3. Find clean gloves in the bags at the dedicated eDNA freezer. The gloves do not need to be bleached since the bags/room are “dirty”. 4. Find the desired bag of filters to extract in the eDNA freezer. Extraction Protocol – Sterivex filters 2 5. When entering the air lock in room 358, switch gloves. Spray gloves with bleach. Spray the outside of bags liberally with 10% bleach. Allow the sample bag to sit w/ bleach for 5 minutes, then dry them. 6. Place the bags in one of the dedicated plastic boxes with lid in the fridge in the air-lock at 4oC for gentle thawing. It takes approx. 1-2 hours. 7. Add the samples to the eDNA sample overview file DAY 1: 1. Follow the descriptions in the ‘Clean Lab Routines’ of how to enter the labs. 2. Clean the bags containing the samples very thoroughly using 10% bleach, water and then 70 %EtOH. 3. Bring these cleaned bags into the clean lab following the clean lab routines. 4. To remove excess water inside the filters, place the inlet of the filter (narrow end) in a 1.5 ml Eppendorf tube and gently slide filter and tube into the 50 ml falcon tube that contained the filter (or in a new 50 ml tube if samples were stored in ziplock bags). If more than one filter is in the tube, label a new tube for the second filter. When done with filters from one species/station, clean everything again (forceps, gloves, working surface) with bleach, MilliQ water and ethanol, before proceeding to the next species/station. 5. Centrifuge the tubes at 1500 x g for 3 minutes to remove the remaining seawater from the filters. 6. Make extraction buffer solution for adding 2.5X the recommended volume = 500µl per filter. o Recommended volume is 20µl Proteinase K + 180µl Buffer ATL per sample: ▪ 2.5 * 20µl ProK = 50µl ▪ 2.5 * 180µl ATL = 450 ul ▪ Total amount of extraction buffer per sample = 500µl ▪ E.g. for 20 samples: 1000µl ProK, 9000µl ATL. First, pipette the 9ml with a sterile glass pipette into a clean 50ml falcon tube. Then pipette 1 ml of ProK into the same tube. Close with lid and invert solution, avoiding foaming. 7. Add 500µl of the extraction solution to each filter, starting with blanks, by pushing the 1000µl tip tight into the outlet end of the filter and gently aspirating the solution into the filter. Take care that all the solution goes into the filter. If the filter is clogged, then aspirate from the inlet end of the filter. 8. Cap the filters with sterile caps. Make sure that its completely sealed. 9. MAKE SURE YOU LABEL ALL THE FILTERS CORRESPONDING TO THE TUBES, by writing the label and the replicate letter (A, B, C etc.) on the filter and cover with tape. 10. Place the filters in rotator and fasten them with the elastic band. 11. When done with all filters, move the rotator to the incubator oven (56C). Make sure that the rotator is moving at 6 rpm and not hitting the oven. Check the filters after a couple of hours and leave them overnight for the 2nd day of extractions. Minimum 8-12 hours incubation. 12. Note: Always use similar incubation time for all filters within a project. Note the time for when incubation in the incubator oven started. DAY 2 13. Enter lab and clean according to the Clean Lab Routines. 14. Label all tubes needed for the process: 2ml Eppendorf tubes, spin columns and the final 1.5ml Eppendorf tubes that will hold the eluted DNA (sample ID on top, and more details on the side including replication (A,B,C), depth, date of collection, date of extraction and your initials). 15. Note the time when the filters are removed from the incubator oven. 16. Reopen the sealed filters and transfer them to a marked 2ml tube inside a new 50ml falcon tube with the inlet facing down into the 2ml Eppendorf tube. 17. Centrifuge the 50ml tubes containing the 2ml tubes and the filters at 1700 x g for 3 minutes. 18. Remove the filter from the 50ml tube and discard it. Then carefully remove the 2ml tube from the bottom of 50ml tubes with a tweezer holding the root of the cap, without touching the cap itself or the edge of Extraction Protocol – Sterivex filters 3 the tube opening. Close the 2ml tube and place it in a rack. Again, start with the lowest concentration (e.g. air-> blank -> real samples). 19. “Measure” the approximate volume of 2-3 samples using a pipette with NEW tips for each sample. Round the mean volume to nearest 50µl. 20. Add an equal volume of the Buffer AL as the one determined above (7.) and ensure to mix it with the pipette immediately using new tips for each sample. 21. Add an equal volume of 100% EtOH as the one determined above (7.) and ensure to mix it with the pipette immediately using new tips for each sample. 22. Vortex and spin down the samples to make sure it is mixed and liquid from the cap is removed. 23. Place the spin columns in front of the samples in the rack. 24. Transfer 630µl of the sample into corresponding spin column. Be careful not to make any bubbles but at the same time try not to leave liquid in the tip because it is precious DNA. 25. Centrifuge the columns at 15.000 x g for 2 mins. 26. Discard the collection tube with the flow-through and transfer the spin column to a new collection tube. Make sure the flow-through has not spilled back to the column when you removed it from the centrifuge. 27. Transfer the rest of the sample to the corresponding spin column. If more than 630µl, three rounds of spinning are required. 28. Centrifuge the columns at 15.000 x g for 2 mins. 29. Discard the collection tube with the flow-through and transfer the spin column to a new collection tube. Make sure the flow-through has not spilled back to the column when you removed it from the centrifuge. 30. Add 500µl Buffer AW1 (check EtOH has been added to buffer) using new tips for each tube. 31. Centrifuge at 15000 x g for 2 mins. 32. Discard the collection tube with the flow-through and transfer the spin column to a new collection tube. Make sure the flow-through has not spilled back to the column when you removed it from the centrifuge. 33. Add 500µl Buffer AW2 and centrifuge for 4 mins at 20.000 x g. 34. While centrifuging, clean flowhood, pipettes, and pens with bleach, MilliQ and ethanol. 35. TAKE GREAT CARE that no flow-through is present on the sides of the spin columns. If so, spin the columns again in a new collection tube at 20.000 x g for 2 mins. Note what samples that have been centrifuged twice. 36. Transfer the spin-columns to the corresponding Eppendorf tubes. Make sure that the lid/tap of the spin column does not touch the cap of the Eppendorf tube to avoid contamination. 37. Add 75µl of Buffer AE to each spin columns. Make sure to add the buffer at the center of the membrane without touching the membrane. Incubate for 1 min, then spin the samples at 20.000 x g for 2 mins. 38. Discard the spin columns and transfer a 20µl aliquot of the extracted DNA from each sample to a PCR plate or PCR strips. It is very important the plate/strip is labeled properly with all necessary information and with unique names (not just ‘Plate 1’!). If you are using strips, use an empty pipette tip box as rack. Wrap aliquots in two bags before temporary storage. Place the aliquot in the fridge at 4C if you are certain it will be processed within the next 2-3 weeks or in the aliquot freezer if longer. 39. Store the rest of the DNA as stock in the freezer located in the extraction lab. Store the 1.5ml tubes in a cryobox that you have purchased at the store and brought with you. Make sure to label the box properly. Put the cryobox in two bags before storing it and ONLY thaw the stock if absolutely necessary. 40. Clean the laminar flow-hood and all equipment according to the clean-lab guidelines.