Cat. No. D636401 / D636402 / D636404
Manual single-tube magnetic bead workflow for microbial DNA purification from stool samples.
Transfer 100–150 mg stool sample to a 2 ml Bead Tube. For liquid stool samples, pipette about 0.15 ml sample; cut the end of the pipette tip if needed to make pipetting easier.
Stool consistency affects pipetting and lysis efficiency. Keep the input within the recommended range to avoid excessive inhibitor load.
Add 0.6 ml Buffer ATL / PVP-10 and 0.6 ml Buffer PCI to the sample. Process on a bead beater machine or vortex at maximum speed for 10 min.
PVP-10 powder should be added into Buffer ATL before use and dissolved completely by inversion. Processing time depends on sample input and bead-beater format.
Incubate the disrupted sample at 65°C for 20 min.
This step supports more complete bacterial lysis after bead-based disruption of the stool matrix.
Centrifuge at 13,000 × g for 5 min to pellet stool debris and inhibitor-rich particulate material.
Clear lysate separation reduces carryover into the magnetic-bead purification stage.
Transfer 400 µl supernatant to a new 2 ml centrifuge tube.
Avoid disturbing the pellet when collecting the clarified fraction.
Add 10 µl RNase A, mix thoroughly and sit at room temperature for 5–10 min.
The timeline uses the midpoint of the short incubation range.
Add 30 µl MagPure Particles N, 20 µl Proteinase K and 600 µl Buffer MLE to the sample. Mix thoroughly by vortexing for 10 min.
Buffer MLE should be prepared with isopropanol as described in the manual. Shake MagPure Particles N vigorously for 1–2 min before use to obtain a homogeneous suspension.
Place the tube on a magnetic stand for 2–3 min until the beads form a tight pellet, then remove the supernatant.
The timeline uses the midpoint of the magnetic separation range plus experienced handling time for supernatant removal.
Add 600 µl Buffer GW1, vortex for 15 s to resuspend beads, place on the magnetic stand for 1 min and remove the supernatant.
Confirm that ethanol has been added to Buffer GW1 before use.
Add 600 µl 75% ethanol, vortex for 15 s to resuspend beads, place on the magnetic stand for 1 min and remove the supernatant.
Efficient ethanol washing helps reduce residual salts and contaminants.
Repeat the 75% ethanol wash once using the same resuspension, magnetic separation and supernatant-removal logic.
Remove the wash liquid carefully to reduce ethanol carryover.
Centrifuge briefly to collect liquid on the tube, place the tube on the magnetic stand and remove all liquid carefully.
This step prepares the beads for controlled air drying.
Air-dry the magnetic beads for 10 min.
Do not leave visible ethanol before elution; incomplete drying can affect downstream PCR or enzymatic reactions.
Add 50–100 µl Elution Buffer, resuspend the beads by vortexing and incubate at 55°C for 10 min with shaking. If no shaking device is available, vortex 2–3 times during incubation.
Warm elution supports DNA release from magnetic particles.
Place the tube on the magnetic rack for 2 min, then transfer the supernatant containing purified DNA to a clean 1.5 ml centrifuge tube.
Avoid transferring magnetic particles into the final eluate.
This workflow separates stool-matrix bead disruption and heat-assisted lysis from RNase treatment, magnetic-particle binding, washing, drying and elution. It is intended as a practical companion to the product manual rather than a replacement for the official protocol. This note reflects the manual single-tube process; the high-throughput process can be followed according to the official protocol when plate-based handling is required.
Protocol times stated in the product manual are retained where applicable. Steps without explicit timing are estimated for an experienced operator, including pipetting, vortexing, tube transfer, magnetic-rack handling, supernatant removal and tube 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. In this workflow, bead disruption is displayed as the 10-minute lysis-by-beads step stated in the manual; actual handling may vary with stool consistency, sample input and bead-beater format. Cumulative time runs continuously from sample loading to final DNA recovery.
D6364 combines stool bead-tube disruption, ATL / PVP-10 / PCI lysis chemistry, RNase treatment and MagPure Particles N purification. The early workflow is designed to release microbial DNA from stool while reducing humic acid and other inhibitory factors before magnetic-bead binding and ethanol-based washing.
The main handling risks are excessive stool input, incomplete PVP-10 dissolution, poor bead-particle resuspension and carryover of inhibitor-rich debris after clarification. Keep the clarified lysate clean before RNase treatment and binding. Ensure that Buffer GW1 contains ethanol and Buffer MLE contains isopropanol before use, and remove residual ethanol carefully before elution. The manual also provides a high-throughput process for plate-based workflows.