Sample input
Transfer 250–500 mg soil, 50–100 mg stool, or 200–500 mg other environmental sample into a 2 ml Bead Tube.
Reduce input for very viscous, organic-rich or inhibitor-heavy material if clogging or dark eluate is expected.
Workflow Note
HiPure Soil DNA Kit — Soil Matrix Workflow Note
Cat. No. D314202 / D314203
Soil and environmental sample DNA extraction using bead disruption, inhibitor adsorption and silica spin-column purification
Matrix disruption and lysis
Inhibitor adsorption
Column purification
2 min
Cumulative 2 min
3 min
Cumulative 5 min
SOL lysis setup and bead disruption
Add 800 μl Buffer SOL. Homogenize by bead beating; the standard timeline assumes a rapid bead-mill route such as two 45 s FastPrep cycles with handling time.
Vortex disruption for 10 min is also supported and will extend the front-end workflow time.
12 min
Cumulative 17 min
SDS-assisted heat lysis
Briefly spin down liquid from the lid, add 60 μl Buffer SDS, mix thoroughly and incubate at 65°C for 10 min.
This step supports detergent-assisted lysis of bacteria, yeast and fungal cells in soil or environmental matrices.
1 min
Cumulative 18 min
Clarify crude lysate
Centrifuge at 13,000 × g for 1 min at room temperature.
A compact pellet helps reduce particulate carryover before inhibitor adsorption.
2 min
Cumulative 20 min
Transfer supernatant
Transfer 600 μl supernatant into a new 1.5 ml microcentrifuge tube.
Avoid disturbing soil particles or bead debris at the bottom of the tube.
5 min
Cumulative 25 min
Inhibitor adsorption
Add 150 μl Buffer PS and mix. Add 150 μl Absorber Solution, mix thoroughly and incubate for 3 min.
Absorber Solution is useful for pigment or humic-inhibitor removal, but may be omitted for very low-nucleic-acid or light-colored samples.
5 min
Cumulative 30 min
Clarify after adsorption
Centrifuge at maximum speed, ≥13,000 × g, for 5 min.
This separates the adsorbed contaminants from the cleared DNA-containing supernatant.
2 min
Cumulative 32 min
Recover cleared supernatant
Transfer the cleared supernatant, usually about 750 μl, to a new 2 ml microcentrifuge tube.
Clean transfer at this point is more important than recovering every remaining microliter.
2 min
Cumulative 34 min
Adjust binding conditions
Add an equal volume of Buffer GWP and mix by inverting the tube 4–6 times.
Accurate volume matching helps establish the correct salt-mediated binding condition for the column.
6 min
Cumulative 40 min
Two-load column binding
Load 750 μl of the mixture onto the HiPure DNA Mini Column II and centrifuge at 10,000 × g for 1 min. Discard flow-through and repeat with the remaining mixture.
Repeated loading is included in the cumulative time because the full binding mixture exceeds a single column load.
4 min
Cumulative 44 min
DW1 wash
Add 500 μl Buffer DW1, incubate for 1 min, centrifuge at 10,000 × g for 1 min and discard the flow-through.
This wash helps remove residual contaminants before ethanol-containing washes.
6 min
Cumulative 50 min
GW2 wash ×2
Wash the column twice with 650 μl Buffer GW2, centrifuging at 10,000 × g for 1 min each time and discarding flow-through between washes.
Confirm that ethanol has been added to Buffer GW2 before use.
2 min
Cumulative 52 min
Dry spin
Discard flow-through and centrifuge the column at 10,000 × g for 1 min to reduce Buffer GW2 carryover.
Residual ethanol can affect downstream enzymatic reactions, so complete drainage is important.
5 min
Cumulative 57 min
First elution
Transfer the column to a 1.5 ml tube. Add 50 μl preheated Buffer AE directly to the membrane, incubate for 3 min and centrifuge at 10,000 × g for 1 min.
Preheated elution buffer supports recovery from the silica membrane.
5 min
Cumulative 62 min
Second elution or re-elution
Add another 50 μl preheated Buffer AE, or reload the eluate onto the membrane, incubate for 3 min and centrifuge at 10,000 × g for 1 min.
Use separate elution for higher total recovery or re-elution when a more concentrated eluate is preferred.
1 min
Cumulative 63 min
Store purified DNA
Discard the column and store purified DNA at −20°C.
Purified DNA is suitable for PCR, restriction digestion and next-generation sequencing.
Typical processing time under routine manual operation≈ 60–70 min
Fast protocol option: The product manual also provides a shortened route in which Buffer SOL and Buffer SDS are added together before disruption, followed by direct clarification and inhibitor adsorption. This can reduce the front-end workflow, but the standard timeline above follows the full soil matrix workflow because it better represents the separate lysis, heat-treatment and inhibitor-removal logic.
How to Read This Note
1. Workflow structure
This workflow separates soil or environmental sample disruption, inhibitor adsorption and the downstream silica spin-column purification path. It is intended as a practical companion to the product manual rather than a replacement for the official protocol. The standard route is shown because it better represents the full soil matrix workflow, including detergent-assisted heat lysis and Absorber Solution treatment before column binding.
2. Time interpretation
Protocol times stated in the product manual are retained where applicable. Steps without explicit timing are estimated for an experienced operator, including pipetting, tube transfer, centrifuge handling, supernatant or flow-through removal and tube or column repositioning. For short protocol ranges, the timeline uses the midpoint. For long or optional protocol ranges, the displayed standard timeline uses the shortest reasonable path, while the note and total-time range indicate where extended handling may apply. Cumulative time runs continuously from sample input to final elution across all workflow sections. Downstream column steps include handling time for repeated loading, buffer addition, flow-through disposal and column repositioning.
3. Workflow characteristics
D3142 is not a simple direct lysis route. Soil and environmental samples may contain humic acid, pigments, polysaccharides, proteins and particulate material that can interfere with PCR, restriction digestion or sequencing. The workflow first releases DNA through bead disruption and detergent-assisted lysis, then uses Buffer PS and Absorber Solution to reduce inhibitor load before salt-mediated DNA binding on the silica column.
4. Practical considerations
The key handling point is clean separation of clarified supernatant from soil debris and adsorbed contaminants. Absorber Solution is useful for colored or inhibitor-rich lysates, but the manual notes that it may also adsorb a small amount of nucleic acid; for low-biomass or light-colored samples, omission may be considered according to the official protocol. Excess starting material, viscous lysate or insufficient disruption increases the risk of column clogging, dark membrane staining or colored eluate.