SPL / PS clarification and magnetic-particle purification for plant and fungal DNA
Cat.No. D635100 / D635101 / D635102 / D635103 · Manual workflow overview
Disrupt plant or fungal tissue by liquid-nitrogen grinding or another bead-beat method. Transfer up to 100 mg wet tissue or 20 mg dried tissue into an appropriate tube and avoid thawing before lysis.
For difficult samples, first optimize the sample amount before changing the chemistry.
Add 500 µl Buffer SPL and 10 µl RNase Solution. Vortex vigorously and pipet if needed until visible clumps are dispersed.
Do not premix Buffer SPL and RNase Solution before use.
Incubate at 65°C for 10 minutes. Invert 2–3 times during incubation so the disrupted material remains in contact with the lysis buffer.
RNase digestion occurs during the lysis stage, before magnetic-particle binding.
Add 170 µl Buffer PS, vortex to mix, and incubate for 5 minutes on ice.
Buffer PS is used to help remove cell debris, precipitated proteins and polysaccharides before magnetic binding.
Centrifuge the lysate for 5 minutes at >13,000 × g. Keep the pellet compact and visible before supernatant transfer.
The DNA-containing fraction used for purification is the clarified supernatant, not the cell-debris pellet.
Transfer 500 µl of the supernatant to a clean 1.5 ml centrifuge tube without disturbing the cell-debris pellet.
Carryover of pellet material can increase viscosity and reduce magnetic-particle performance.
Add 30 µl MagPure Particles and 350 µl 100% ethanol. Mix by pipetting 20 times or shaking for 5 minutes until the liquid appears homogeneous.
Shaking for 5 minutes may improve yield compared with pipette mixing alone. MagPure Particles should be fully resuspended before dispensing.
Place the tube on a magnetic stand for 2 minutes until the particles form a tight pellet. Remove the supernatant carefully.
After binding, target DNA is on the magnetic particles; do not discard or aspirate the bead pellet.
Add 500 µl Buffer GW1, resuspend the beads by shaking for 1 minute, place on the magnetic stand for 1 minute, and remove the supernatant.
Ensure that ethanol has been added to Buffer GW1 before use.
Repeat the GW1 wash once to further remove soluble contaminants and residual binding components.
Complete bead resuspension is more important than simply adding the wash buffer.
Add 600 µl 80% ethanol, resuspend the beads by shaking for 1 minute, place on the magnetic stand for 1 minute, and remove the supernatant.
This wash helps remove salts while keeping the DNA associated with the particles.
Add 600 µl absolute ethanol, resuspend the beads by shaking for 1 minute, place on the magnetic stand for 1 minute, and remove the supernatant.
Remove the ethanol wash as completely as practical before the drying step.
Air-dry the particle pellet for 10 minutes.
Insufficient drying can leave ethanol in the eluate; excessive drying can make the pellet difficult to resuspend.
Add 50–100 µl Elution Buffer, resuspend the particles by vortexing, and incubate at 65°C for 10 minutes with shaking. If no shaking device is available, vortex 2–3 times during incubation. Place on the magnetic rack for 2 minutes.
Elution depends on complete particle resuspension and adequate contact between Elution Buffer and bead-bound DNA.
Transfer the supernatant containing purified DNA to a clean 1.5 ml centrifuge tube.
Avoid carrying magnetic particles into the final eluate.
This workflow follows the manual plant and fungal tissue route for MagPure Plant DNA Kit. It separates the front-end disruption, SPL lysis and PS clarification stage from the shared magnetic-particle binding, wash and elution route. It is intended as a practical companion to the product manual rather than a replacement for the official protocol.
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, magnetic-rack placement, bead resuspension, supernatant removal, residual-liquid removal, drying control, elution and final tube transfer. For short protocol ranges, the timeline uses the midpoint or a near-midpoint value. 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. For this D6351 workflow, the displayed timeline represents a standard manual run and ends at 75 min. Because the core timing anchors are relatively fixed, the final time range is kept close to the timeline; the remaining variation mainly comes from sample disruption quality, lysate viscosity, bead resuspension, complete wash-solution removal and final 65°C elution handling.
D6351 is a magnetic plant DNA workflow. Buffer SPL lyses mechanically disrupted plant or fungal material while RNase Solution acts during lysis. Buffer PS helps remove cell debris, precipitated proteins and polysaccharides. DNA in the clarified supernatant is then bound to MagPure Particles in the presence of ethanol, washed with GW1, 80% ethanol and absolute ethanol, dried, and eluted with Elution Buffer at 65°C.
Do not exceed the recommended input amount, and fully disperse the sample powder in SPL before lysis. After PS treatment and centrifugation, transfer the clarified supernatant without disturbing the cell-debris pellet. MagPure Particles must be homogeneous before dispensing, and the beads must be fully resuspended during binding, washing and elution. For complicated samples, 2-mercaptoethanol may be added to Buffer SPL at 2% (v/v), equivalent to 20 µl per 1 ml Buffer SPL. The manual recommends optimizing sample amount first; for dry or polysaccharide-rich samples, CTAB lysis with chloroform extraction may be considered as a sample-specific optimization instead of the standard SPL / PS extraction stage.