Nov 14, 2023
Genomic DNA extraction from STERIVEX filter within capsule using QIAGEN´s DNeasy Blood and Tissue kit
This protocol is a draft, published without a DOI.
- 1Universidad de Aysén
Protocol Citation: delphine.vanhaecke 2023. Genomic DNA extraction from STERIVEX filter within capsule using QIAGEN´s DNeasy Blood and Tissue kit. protocols.io https://protocols.io/view/genomic-dna-extraction-from-sterivex-filter-within-c4ysyxwe
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 13, 2023
Last Modified: November 14, 2023
Protocol Integer ID: 90866
Keywords: eDNA extraction, sterivex
Funders Acknowledgement:
Gobierno Regional de Aysén
Grant ID: FIC 2022 codigo BIP 40049315-0
Abstract
This is our protocol for extracting environmental DNA from STERIVEX filters using QIAGEN´s Dneasy Blood and Tissue KIT. The protocol is a combination of previously described protocols from:
DNeasy Blood & Tissue Handbook (June 2023). QIAGEN
Kawato, M., Yoshida, T., Miya, M., Tsuchida, S., Nagano, Y., Nomura, M., Yabuki, A., Fujiwara, Y., & Fujikura, K. (2021). Optimization of environmental DNA extraction and amplification methods for metabarcoding of deep-sea fish. MethodsX, 8, 101238. https://doi.org/https://doi.org/10.1016/j.mex.2021.101238
Spens, J., Evans, A. R., Halfmaerten, D., Knudsen, S. W., Sengupta, M. E., Mak, S. S. T., Sigsgaard, E. E., & Hellström, M. (2017). Comparison of capture and storage methods for aqueous macrobial eDNA using an optimized extraction protocol: advantage of enclosed filter. Methods in Ecology and Evolution, 8(5), 635-645. https://doi.org/https://doi.org/10.1111/2041-210X.12683
Wong, M. K.-S., Nakao, M., & Hyodo, S. (2020). Field application of an improved protocol for environmental DNA extraction, purification, and measurement using Sterivex filter. Scientific Reports, 10(1), 21531. https://doi.org/10.1038/s41598-020-77304-7
Materials
DNeasy Blood and Tissue kit for DNA extraction (QIAGEN)
Laminar flow hood with UV
2ml Luer Lock syringes
Petri dishes
Micropipets and tips
Incubator
Microcentrifuge
Pre and post PCR rooms
Clean the laminar flow hood surface and pipettes with DNAZAP and rinse with ddH2O.
10m
Place gloves and 1.5 mL low bind tubes (for receiving DNA extract elution 1, elution 2 and one DNA extraction control) in the flow hood and expose to UV for 10 minutes.
10m
While exposing to UV in step 2 in the PRE-PCR room, in the POST-PCR room remove the parafilm from the sterivex filters and clean the outside of the sterivex filters with 10% bleach. Let them dry standing in a rack. Label the capsules with the label written on the sterile WhirlPack bag corresponding to each filter.
15m
Heat the incubator to 56*C.
1m
Remove the red cap from the filter and remove the buffer EtOH 100% with a sterile 2mL Leur Lock syringe (do not backflush) and leave it in a sterile 2mL LoBIND tube. Label the tube and store at -20*C or -80*C. (see other protocol for extracting eDNA from EtOH buffer)
20m
Blot the sterivex filter gently on blotting paper to remove excess EtOH 100%.
5m
Prepare in a sterile 50ml eppendorf tube the lysis buffer mix of 990 µl of freshly prepared 1x PBS, 910 µl buffer AL (no EtoH added), 100 µl proteinase K per sample and inject it into the sterivex capsule using a sterile 2mL Luer Lock syringe and backflush (flush the capsule in reverse direction). Close with the red cap, secure both sides with parafilm and place the capsule in a sterile petri dish.
20m
Handshake or vortex the sterivex filter for 15s
5m
Incubate at 56*C for 2 hours and handshake/vortex in between.
2h
Handshake or vortex the filters for 15s
5m
Remove and discard the parafilm and red cap carefully. Remove the lysis buffer mix using a sterile 2mL Luer Lock syringe, backflush, and put it into an sterile 1.5 mL Lobind eppendorf tube.
20m
Discard the sterivex filter.
Add 1:3 EtOH 100% to the lysis buffer mix in the 1.5 mL LoBind tube. (eg if final lysis mix in tube is 1mL than add 333 µl EtOH 100%).
5m
Vortex well for 5s
5m
Pipet max. 600 µl of the mixture into the DNeasy Spin Column placed in a 2ml collection tube. Centrifuge at 8000rpm for 1 min.
5m
Discard flow-through. Repeat this step until the entire mixture has passed through the filter. Discard flow through and collection tube.
5m
Place the DNeasy Spin Column in a new 2 ml collection tube and add 600 µl of Buffer AW1. Centrifuge at 8000rpm for 1min. Discard flow through and collection tube.
5m
Place the DNeasy Spin Column in a new 2 ml collection tube and add 600 µl of Buffer AW2. Centrifuge at 14.000rpm for 3min to dry the DNEasy membrane. Discard flow through and collection tube.
10m
Place the DNeasy Spin Column in a sterile 1.5 ml LoBIND eppendorf tube and pipet 75 µl of buffer AE directly onto the DNeasy membrane. Incubate at room temperature for 1min and centrifuge at 8000rpm for 1min to elute DNA.
5m
Repeat step 18 in order to obtain a second elution of the DNA sample.
5m
Aliquote 30µl of the DNA samples for further analysis (QUBIT and PCR) and store at 4*C. Store the stock DNA samples at -80*C.
15m