Magnetic-particle plant and fungal RNA workflow with integrated DNase I treatment
Cat.No. R664101 / R664102 / R664103. Manual 96-well magnetic-plate workflow for plant and fungal sample lysates.
Use the appropriate plant or fungal input and grind the sample thoroughly under liquid nitrogen. Transfer the powder to a 2 ml microcentrifuge tube, allow liquid nitrogen to evaporate, and do not allow the sample to thaw before lysis.
For plant material, start with no more than 30 mg where possible and do not exceed 50 mg. Complete disruption and rapid buffer contact are critical for RNA recovery.
Add 600 µl Buffer PRC1 or Buffer RL to up to 50 mg tissue powder. Vortex vigorously to disperse the sample and centrifuge for 5 min at ≥14,000 × g.
Buffer RL is the general lysis buffer. Buffer PRC1 should be selected when secondary metabolites or sample chemistry cause solidification or poor lysate behavior. Optional 2-mercaptoethanol or DTT may be added to PRC1 / RL before use according to the manual.
Add 500 µl Buffer MCB and 30 µl MagPure RNA Particles to the well of a 2.2 ml 96-well plate. Transfer 500 µl clarified lysate supernatant from the centrifuged sample into the binding well.
MagPure RNA Particles must be fully resuspended before dispensing. Avoid transferring pellet material or insoluble plant debris into the binding well.
Pipette-mix the sample about 10 times, then shake at 700–900 rpm for 10 min until the liquid appears homogeneous.
Uniform particle suspension during binding is essential. Poor mixing can reduce RNA recovery and increase well-to-well variation.
Place the deep-well plate on a magnetic plate for 2 min until the particles form a tight pellet. With the plate on the magnet, aspirate and discard the supernatant.
Do not disturb or remove the particle pellet. Particle loss at this stage directly reduces RNA yield.
Add 600 µl Buffer MW1 and shake at 900–1200 rpm for 2 min to resuspend the particles. Place the plate on the magnetic plate for 1 min, then remove the supernatant.
Confirm that Buffer MW1 has been diluted with ethanol before use. The pre-DNase wash helps remove lysate components before nuclease treatment.
Leave the plate on the magnetic plate for 1 min. Remove residual liquid with a pipettor and dry the MagPure RNA Particles for an additional 3 min.
Remove visible residual wash liquid, but avoid excessive drying before DNase treatment because the particles must be efficiently resuspended in DNase mixture.
Add 300 µl DNase mixture to each sample, prepared from 290 µl DNase Buffer and 10 µl DNase I. Mix by shaking at 600–900 rpm for 10–15 min.
The displayed time includes preparation/addition of DNase mixture plus a near-midpoint digestion period. Complete particle resuspension is required for effective DNA removal.
Add 450 µl Buffer MCB to the DNase-treated sample and shake for 5 min. Place the plate on the magnetic plate for 1 min, then remove the supernatant.
This step restores binding conditions after DNase treatment so RNA remains associated with the magnetic particles during subsequent washing.
Add 600 µl Buffer MW1 and shake for 1 min to resuspend the particles. Place the plate on the magnetic plate for 1 min, then remove the supernatant.
Efficient MW1 washing removes residual DNase reaction components and binding contaminants.
Add 600 µl Buffer MW2 and shake for 1 min to resuspend the particles. Place the plate on the magnetic plate for 1 min, then remove the supernatant.
Confirm that Buffer MW2 has been diluted with ethanol before use.
Repeat the MW2 wash once using 600 µl Buffer MW2, followed by magnetic separation and supernatant removal.
The second ethanol-containing wash improves salt and contaminant removal before final drying.
Leave the plate on the magnetic plate for 1 min, remove residual liquid with a pipettor, and dry the MagPure RNA Particles for an additional 10 min.
Residual ethanol can inhibit downstream RT-PCR. Avoid both visible carryover and excessive over-drying, which can make particles difficult to resuspend during elution.
Add 50–100 µl RNase Free Water to each sample and shake for 5 min. Place the plate on the magnetic plate for 3 min, then transfer the purified RNA-containing supernatant to a new plate or tube.
Store purified RNA at −80°C for long-term storage. Avoid transferring magnetic particles with the eluate.
This note follows the R6641 manual magnetic-particle route: liquid-nitrogen disruption, PRC1 / RL lysis and clarification, magnetic RNA binding with Buffer MCB and MagPure RNA Particles, MW1 wash, on-bead DNase I treatment, MCB rebinding, MW1 / MW2 washing, drying and RNase-free water elution. 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, plate handling, centrifuge handling, magnetic-rack placement, bead resuspension, supernatant removal, residual-liquid removal, drying control, elution and final tube or plate 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 R6641 workflow, the main timing variables are liquid-nitrogen grinding, lysate clarity, particle resuspension, magnetic separation behavior, residual-liquid removal and the 10–15 min DNase mixing window.
R6641 uses high-binding magnetic RNA particles in a plate-based workflow. Buffer RL is used as the general lysis buffer, while Buffer PRC1 is selected when plant secondary metabolites cause poor lysate behavior. DNase I treatment is integrated after initial RNA binding and a pre-DNase MW1 wash, followed by MCB rebinding before the final wash sequence. The automation route is described in the product manual; this note focuses on the manual 96-well magnetic-plate workflow.
Use an appropriate plant input and avoid exceeding 50 mg per preparation. Keep tissue frozen until lysis buffer is added, fully disperse the powder in PRC1 / RL, and transfer only clarified supernatant into the binding well. Resuspend MagPure RNA Particles completely at every binding, washing, DNase and elution step. Confirm that Buffer MCB, MW1 and MW2 have been prepared with the specified alcohols before use. During magnetic separation, avoid particle loss; during drying, remove visible residual liquid without over-drying the beads.