Nov 04, 2024
SDS-based DNA extraction method of Sediment
- Qianyue Ji1,
- Fangfang Jiang2,
- Xiaohan Wang1,
- Shanshan Liu3,
- Mo Han1
- 1BGI research;
- 2China National GeneBank;
- 3BGI
Protocol Citation: Qianyue Ji, Fangfang Jiang, Xiaohan Wang, Shanshan Liu, Mo Han 2024. SDS-based DNA extraction method of Sediment . protocols.io https://dx.doi.org/10.17504/protocols.io.14egn9bypl5d/v1
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: October 27, 2024
Last Modified: November 04, 2024
Protocol Integer ID: 110975
Abstract
This method presents an optimized protocol of SDS-based DNA extraction method for sediment samples.
The extracted DNA can be used for metagenomic research.
Materials
Materials
MGIEasy Stool Microbiome DNA Extraction Kit (940-000122-00, MGI Tech)
Magnetic bead purification (N411-03, Vazyme) or Column purification (47014, QIAGEN)
Supplies:
- Pipette tips (assorted volumes)
- Tubes
Equipment:
- Micropipettes, various volumes
- Microcentrifuge
- Vortex
- MGISP-NE384, MGI Tech
- Magnetic rack
Sample preparation
Sample preparation
Sample preparation
10 g sediment samples were firstly split into aliquots of 1 g to adapt the 2 mL centrifuge tubes and high-speed centrifuge.
After removal of the supernatants all aliquots of each sample were combined.
DNA Extraction
DNA Extraction
DNA Extraction
10 mL lysis solution (0.1 Molarity (M) NaH2PO4, 0.1 Molarity (M) Na2HPO4, 0.1 Molarity (M) EDTA, 0.1 Molarity (M) Tris-HCl, 1.5 Molarity (M) NaCl, 1 % (v/v) CTAB) were added to each sample.
The samples were shaken for 00:05:00
5m
100 µL lysozyme (100 % (v/v) ) and 100 µL proteinase k (20 % (v/v) ) were added to the sample.
Incubation at 37 °C for 00:30:00 .
30m
870 µL 20 % (v/v) SDS was added to the sample, followed by incubation at 65 °C for 02:00:00
2h
Chloroform extraction
Chloroform extraction
Chloroform extraction
The samples were centrifuged for00:10:00 at 10000 x g, 25°C , and collect supernatant to a new 2 mL centrifuge tube.
10m
The supernatant was extracted twice with equal volume mixture of phenol, chloroform, and isoamyl alcohol (25:24:1 v/v/v).
Isopropanol precipitation
Isopropanol precipitation
isopropanol precipitation
DNA was precipitated overnight at -20 °C with 0.6× volume of isopropanol and 0.1× volume of 3M NaAc (5.2 )
The precipitated DNA was washed twice with 70 % (v/v) 70% ethanol, and finally resuspended in 100 µL TE buffer (8 ).
DNA Purification
DNA Purification
26m 30s
26m 30s
Two different methods, column purification (step 6) or magnetic bead purification (step 7), were employed to purify the manually extracted DNA.
Column purification (47014, QIAGEN)
Load DNA sample onto an MB Spin Column and centrifuge at 15.000 x g for 00:01:00 .
1m
Discard the flow-through and repeat step 6.1 to ensure that all of the lysate has passed through the MB Spin Column.
Carefully place the MB Spin Column into a clean 2 ml Collection Tube. Avoid splashing any flow-through onto the MB Spin Column.
Add 500 µL of Solution EA to the MB Spin Column. Centrifuge at 15.000 x g for00:01:00 .
1m
Discard the flow-through and place the MB Spin Column back into the same 2 ml Collection Tube.
Add500 µL of Solution C5 to the MB Spin Column. Centrifuge at 15.000 x g for 00:01:00 .
1m
Discard the flow-through and place the MB Spin Column into a new 2 ml Collection Tube.
Centrifuge at up to 16.000 x g for 00:02:00 . Carefully place the MB Spin Column into a new 1.5 ml Elution Tube.
2m
Add 100 µL of Solution C6 to the center of the white filter membrane.
Centrifuge at 15.000 x g for00:01:00 . Discard the MB Spin Column.
1m
Magnetic bead purification (N411-03, Vazyme)
Mix the DNA Clean Beads thoroughly. Add of 2X volume of DNA Clean Beads to each sample tube. Mix with a vortexer until all beads are suspended.
Incubate at Room temperature for00:05:00 .
5m
Centrifuge the tube(s) briefly and place on the magnetic rack for00:05:00 until the liquid is clear. Carefully remove and discard the supernatant.
5m
While keeping the tube(s) on the magnetic rack, add 500 µL of 80% ethanol to each tube to wash the beads and tube wall. Wait for00:00:30 . Carefully remove and discard the supernatant.
30s
Repeat step7.4. Try to remove all liquid from the tube. If some liquid remains on the tube wall, centrifuge the tube briefly and place it on the magnetic rack for separation. Remove all liquid by using a low-volume pipette.
Keep the tube(s) on the magnetic rack. Open the tube cap and air-dry the beads at room temperature until no wetness or glossiness is visible on the beads' surface. There should be no visible cracking on the surface of the beads.
Remove the tube(s) from the magnetic rack and add 50 µL of TE Buffer to elute the DNA. Mix with a vortexer until all beads are suspended.
Incubate at Room temperature for00:05:00 .
5m
Centrifuge the tube briefly and place on the magnetic rack for 00:05:00 until the liquid is clear. Carefully transfer 48 µL of supernatant to a new 1.5 mL centrifuge tube.
5m