Mar 23, 2023

Public workspaceSuperSoil - Soil DNA Extraction

DOI
dx.doi.org/10.17504/protocols.io.4r3l27864g1y/v1
  • 1National Chung Hsing University, Taichung;
  • 2Kaohsiung Medical University
Jie Hao Ou
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DOI: dx.doi.org/10.17504/protocols.io.4r3l27864g1y/v1
Protocol Citation: Jie Hao Ou, Yin-Tse Huang 2023. SuperSoil - Soil DNA Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l27864g1y/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: September 26, 2022
Last Modified: September 13, 2023
Protocol Integer ID: 70491
Keywords: Soil DNA extraction
Abstract
SuperSoil - Soil DNA Extraction
Guidelines
Keep all solutions at room temperature
Materials
1. Bead (ball mill)
AB
Zirconia Balls150 µm : 500 µm = 1 : 2
2. Solution SD1 (Chaotropic)

ABC
(w/w)Price
Sodium thiocyanate5%1400/250 g
Na2HPO42.5%1350/500 g
pH~8.4
3. Solution SD2

ABC
(w/w)Price
AlCl3 · 6H2O1.5%900/500 g
Ammonium acetate25%820/ 500 g
pH (Adjust with acetic acid)6.3
4. Solution SD3 (Chaotropic)

ABC
(w/w)Price
Guanidinium thiocyanate65%1800/250 g
pH~7.0
5. Solution SEA (Wash buffer 1)

ABC
(w/w)Price
Ethanol50%1000/4000 mL
Isopropanol10%150/ 500 g
Guanidine hydrochloride30%2900/1000 g
6.Solution S5 (Wash buffer 2)

ABC
(w/w)Price
Ethanol70%1000/4000 mL
7.Solution S6 (Elution buffer)

ABC
(w/w)Price
Tris Base0.12%3300/1000 g
pH (Adjust with HCl)8

Safety warnings
Some of the chemicals in this protocol may produce hazardous gases if mixed with bleach.
Lysis
Lysis
Add following materials to 1.8 ml centrifuge tube
  1. 250 mg soil
  2. 1.5 g Bead
  3. 800 µl SD1
Vortex for Duration00:10:00 at max speed

Note
Vortex horizontally and ensure the cap is tightly sealed

10m
Centrifigation14000 rpm, 00:01:00

1m
Transfer the supernatant to a clean 1.5 ml or 2 ml tube

Note
Expect 500–600 µl

Remove inhibitor
Remove inhibitor
1m
1m
Add 200 ul SD2
Note
White precipitate forms.
If too much AlCl3 (SD2) is added, it will cause all the DNA to be precipitated along with the inhibitory substances.
See Mustafa (2017) for more info.
CITATION
Irfan Mustafa, Hadiatullah and Sustiyah (2017). Removal of humic acid from peat soils by using AlCl3 prior to DNA extraction. AIP Conference Proceedings.
LINK
doi.org/10.1063/1.4983434


Vortex for Duration00:00:05 at max speed

5s
Centrifigation14000 rpm, 00:01:00

Note
Don't worry if the supernatant is yellow.

1m
Transfer the supernatant to a clean 1.5 ml or 2 ml tube
Note
Expect 500–600 µl

Bind DNA to silica membrane
Bind DNA to silica membrane
2m 5s
2m 5s
Add 600 µl SD3
Vortex for Duration00:00:05 at max speed

5s
Transfer 650 ul lysate to spin column
Centrifigation14000 rpm, 00:01:00 , discard flow-through

1m
Transfer remaining lysate to spin column
Wash silica membrane
Wash silica membrane
1m
1m
Add 500 ul SEA to spin column
Centrifigation14000 rpm, 00:01:00 , discard flow-through
1m
Add 500 ul S5 to spin column
Centrifigation14000 rpm, 00:01:00 , discard flow-through
1m
Centrifigation14000 rpm, 00:01:00 , Centrifuge again to remove remaining ethanol (S5) , discard flow-through
1m
Elution
Elution
Add 50–100 ul S6