Jun 17, 2020
Protocol for DNA extraction from clay-rich subsoils
- 1Georg-August Universität Göttingen
Protocol Citation: Lukas Beule 2020. Protocol for DNA extraction from clay-rich subsoils. protocols.io https://dx.doi.org/10.17504/protocols.io.bhmjj44n
Manuscript citation:
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 in our group and it is published as Guerra et al. 2020 Microorganisms, 8(4), 532; https://doi.org/10.3390/microorganisms8040532
Created: June 17, 2020
Last Modified: June 17, 2020
Protocol Integer ID: 38283
Protocol source
Protocol source
This protocol is the Supplementary File S1 of Guerra et al., Microorganisms 2020,8(4), 532; https://doi.org/10.3390/microorganisms8040532
please cite as Guerra et al. (2020) Microorganisms,8(4), 532.
Sample preparation
Sample preparation
Weigh in 200 ± 5 mg freeze-dried soil into a 2-mL tube.
Subsoil DNA extraction
Subsoil DNA extraction
3h
3h
1. Homogenize the sample by bead beating using 3 tungsten carbide beads (ø 3 mm) for 1 min at 25 Hz using a
swing mill (e.g. MM 400, Retsch, Germany).
2. Add 250 µL PB buffer1 with 0.5% SDS (w/v) and vortex for 10 sec at max speed.
3. Incubate the suspension at 65°C for 10 min and shake the samples every 60 sec for 5 sec.
4. Centrifuge for 1 min at 10,000 rpm and transfer 90 µL of the supernatant into a new 2-mL tube (supernatant will
contain bubbles).
5. Add 810 µL ddH2O (1:10 dilution) and subsequently 900 µL phenol2 and shake the samples briefly.
6. Centrifuge for 10 min at 10,000 rpm and transfer 800 µL of the upper phase into a new 2-mL tube.
7. Add 800 µL chloroform:isoamylalcohol (24:1 (v/v)), incubate 10 min on ice and centrifuge for 10 min at 10,000
rpm.
8. Transfer 700 µL of the upper phase into a new 1.5 mL-tube.
9. Add 700 µL chloroform:isoamylalcohol (24:1), incubate 10 min on ice and centrifuge for 10 min at 10,000 rpm.
10. Transfer 600 µL of the upper phase into a new 1.5 mL-tube containing 200 µL 30% PEG 6000 and 100 µL 5M
NaCl.
11. Shake the mixture and incubate at room temperature for 20 min.
12. Centrifuge for 15 min at max. speed.
13. Wash the pellet with 500 µL 80% EtOH (v/v) twice and centrifuge for 5 min at max speed in-between each
washing before discarding the supernatant. Supernatant should be removed by pipetting.
14. Dry the pellet using vacuum centrifugation at 30°C for 20 minutes or until it is dry under a sterile bench.
15. Add 50 µL TE buffer, vortex or flick the samples to release the pellet from the tube wall and incubate at 42°C for
2h (e.g. in a water bath).
Notes addressing the solutions: 1 PB buffer: 1 M Na2HPO4 and 1 M NaH2PO4, blended to achieve pH 7.2.2 Phenol: redistilled, in TE buffer equilibrated, pH 7.5 to 8.0
3h