Cat. No. R6622 | Manual magnetic-bead total RNA workflow
For animal tissue, plant tissue, cultured cells, yeast cells and bacterial cells. This note summarizes the manual workflow; automation setup is described separately in the product manual.
Use no more than 20 mg animal tissue. Disrupt and homogenize the sample, add 600 µl Buffer RTL, mix thoroughly and centrifuge at 14,000 × g for 3 min at room temperature.
Use the clarified lysate or supernatant for purification. Incomplete disruption or sample overload increases viscosity and reduces magnetic handling efficiency.
Transfer 500 µl lysate or clarified supernatant to a clean 1.5 ml tube.
For tissue, plant, yeast and bacterial samples, avoid transferring visible debris or glass beads into the purification tube.
Add 500 µl Buffer MCB, 30 µl MagPure RNA Particles and 20 µl Proteinase K. Mix up and down 20–30 times, incubate at room temperature for 10 min, mix several times, place on the magnetic rack for 1 min and remove the supernatant.
Buffer MCB must be supplemented with isopropanol before use. Resuspend the particles evenly before pipetting.
Add 600 µl Buffer MW1 and vortex for 20 sec to resuspend the particles. Place on the magnetic rack for 1 min, remove the supernatant, briefly spin down residual liquid, remove it carefully and air-dry for 2 min.
This step reduces carryover before DNase digestion. Do not disturb the bead pellet during residual-liquid removal.
Add 300 µl DNase mixture, prepared from 290 µl DNase Buffer and 10 µl DNase I. Gently shake or vortex to resuspend the particles and incubate at room temperature for 15 min.
DNase contact depends on complete bead resuspension; a compact bead pellet gives uneven digestion.
Add 450 µl Buffer MCB and vortex for 20 sec. Incubate at room temperature for 5 min, mix up and down 2–3 times, place on the magnetic rack for 1 min and remove the supernatant.
This step restores binding conditions after DNase treatment before the final wash sequence.
Add 600 µl Buffer MW1, vortex for 10 sec to resuspend the particles, place on the magnetic rack for 1 min and remove the supernatant.
Complete resuspension improves removal of salts, proteins and digestion carryover.
Add 600 µl Buffer RW2, vortex for 10 sec to resuspend the particles, place on the magnetic rack for 1 min and remove the supernatant.
Buffer RW2 must be supplemented with ethanol before use.
Repeat the RW2 wash once using 600 µl Buffer RW2, then magnetically separate and remove the wash liquid.
The second RW2 wash improves removal of residual salts and alcohol-soluble impurities.
Briefly spin the tube to collect liquid on the wall and cap, place it back on the magnetic rack, remove all residual liquid carefully and dry at room temperature or 37°C for 10 min.
Residual ethanol may inhibit downstream reactions; excessive drying can make the particles difficult to resuspend.
Add 30–100 µl RNase-free Water, vortex to resuspend the particles completely and incubate at room temperature for 3 min.
Use a smaller volume for higher concentration, or a larger volume when recovery is the priority.
Place the tube on the magnetic rack for 3 min. Transfer the supernatant containing purified RNA to a new RNase-free 1.5 ml tube and store at -80°C.
Do not carry magnetic particles into the final RNA eluate.
Disrupt plant sample under liquid nitrogen and transfer up to 50 mg powder to a 1.5 ml tube. Add 600 µl Buffer RTL, mix well by vortexing and centrifuge at 14,000 × g for 3 min at room temperature.
Use the clarified supernatant. Plant debris, polysaccharides or fine powder carryover can increase viscosity and compromise wash efficiency.
Transfer 500 µl lysate or clarified supernatant to a clean 1.5 ml tube.
For tissue, plant, yeast and bacterial samples, avoid transferring visible debris or glass beads into the purification tube.
Add 500 µl Buffer MCB, 30 µl MagPure RNA Particles and 20 µl Proteinase K. Mix up and down 20–30 times, incubate at room temperature for 10 min, mix several times, place on the magnetic rack for 1 min and remove the supernatant.
Buffer MCB must be supplemented with isopropanol before use. Resuspend the particles evenly before pipetting.
Add 600 µl Buffer MW1 and vortex for 20 sec to resuspend the particles. Place on the magnetic rack for 1 min, remove the supernatant, briefly spin down residual liquid, remove it carefully and air-dry for 2 min.
This step reduces carryover before DNase digestion. Do not disturb the bead pellet during residual-liquid removal.
Add 300 µl DNase mixture, prepared from 290 µl DNase Buffer and 10 µl DNase I. Gently shake or vortex to resuspend the particles and incubate at room temperature for 15 min.
DNase contact depends on complete bead resuspension; a compact bead pellet gives uneven digestion.
Add 450 µl Buffer MCB and vortex for 20 sec. Incubate at room temperature for 5 min, mix up and down 2–3 times, place on the magnetic rack for 1 min and remove the supernatant.
This step restores binding conditions after DNase treatment before the final wash sequence.
Add 600 µl Buffer MW1, vortex for 10 sec to resuspend the particles, place on the magnetic rack for 1 min and remove the supernatant.
Complete resuspension improves removal of salts, proteins and digestion carryover.
Add 600 µl Buffer RW2, vortex for 10 sec to resuspend the particles, place on the magnetic rack for 1 min and remove the supernatant.
Buffer RW2 must be supplemented with ethanol before use.
Repeat the RW2 wash once using 600 µl Buffer RW2, then magnetically separate and remove the wash liquid.
The second RW2 wash improves removal of residual salts and alcohol-soluble impurities.
Briefly spin the tube to collect liquid on the wall and cap, place it back on the magnetic rack, remove all residual liquid carefully and dry at room temperature or 37°C for 10 min.
Residual ethanol may inhibit downstream reactions; excessive drying can make the particles difficult to resuspend.
Add 30–100 µl RNase-free Water, vortex to resuspend the particles completely and incubate at room temperature for 3 min.
Use a smaller volume for higher concentration, or a larger volume when recovery is the priority.
Place the tube on the magnetic rack for 3 min. Transfer the supernatant containing purified RNA to a new RNase-free 1.5 ml tube and store at -80°C.
Do not carry magnetic particles into the final RNA eluate.
Harvest no more than 1 × 10⁷ cells. For pelleted cells, loosen the pellet thoroughly and add 500 µl Buffer RTL; for monolayer cells, add 500 µl Buffer RTL directly to the culture dish. Pass the lysate at least 5 times through a blunt 20-gauge needle fitted to an RNase-free syringe.
Needle passage reduces viscosity and improves bead mixing. A viscous lysate should not be carried directly into binding without adequate shearing.
Transfer 500 µl lysate or clarified supernatant to a clean 1.5 ml tube.
For tissue, plant, yeast and bacterial samples, avoid transferring visible debris or glass beads into the purification tube.
Add 500 µl Buffer MCB, 30 µl MagPure RNA Particles and 20 µl Proteinase K. Mix up and down 20–30 times, incubate at room temperature for 10 min, mix several times, place on the magnetic rack for 1 min and remove the supernatant.
Buffer MCB must be supplemented with isopropanol before use. Resuspend the particles evenly before pipetting.
Add 600 µl Buffer MW1 and vortex for 20 sec to resuspend the particles. Place on the magnetic rack for 1 min, remove the supernatant, briefly spin down residual liquid, remove it carefully and air-dry for 2 min.
This step reduces carryover before DNase digestion. Do not disturb the bead pellet during residual-liquid removal.
Add 300 µl DNase mixture, prepared from 290 µl DNase Buffer and 10 µl DNase I. Gently shake or vortex to resuspend the particles and incubate at room temperature for 15 min.
DNase contact depends on complete bead resuspension; a compact bead pellet gives uneven digestion.
Add 450 µl Buffer MCB and vortex for 20 sec. Incubate at room temperature for 5 min, mix up and down 2–3 times, place on the magnetic rack for 1 min and remove the supernatant.
This step restores binding conditions after DNase treatment before the final wash sequence.
Add 600 µl Buffer MW1, vortex for 10 sec to resuspend the particles, place on the magnetic rack for 1 min and remove the supernatant.
Complete resuspension improves removal of salts, proteins and digestion carryover.
Add 600 µl Buffer RW2, vortex for 10 sec to resuspend the particles, place on the magnetic rack for 1 min and remove the supernatant.
Buffer RW2 must be supplemented with ethanol before use.
Repeat the RW2 wash once using 600 µl Buffer RW2, then magnetically separate and remove the wash liquid.
The second RW2 wash improves removal of residual salts and alcohol-soluble impurities.
Briefly spin the tube to collect liquid on the wall and cap, place it back on the magnetic rack, remove all residual liquid carefully and dry at room temperature or 37°C for 10 min.
Residual ethanol may inhibit downstream reactions; excessive drying can make the particles difficult to resuspend.
Add 30–100 µl RNase-free Water, vortex to resuspend the particles completely and incubate at room temperature for 3 min.
Use a smaller volume for higher concentration, or a larger volume when recovery is the priority.
Place the tube on the magnetic rack for 3 min. Transfer the supernatant containing purified RNA to a new RNase-free 1.5 ml tube and store at -80°C.
Do not carry magnetic particles into the final RNA eluate.
Collect about 5 × 10⁶ yeast cells. Add the specified glass beads and 600 µl Buffer RTL, vortex at maximum speed for 10 min and centrifuge at 10,000 × g for 3 min at room temperature.
The 10 min bead-beating step is retained from the manual. Transfer only the clarified lysate and avoid carrying glass beads into the magnetic purification tube.
Transfer 500 µl lysate or clarified supernatant to a clean 1.5 ml tube.
For tissue, plant, yeast and bacterial samples, avoid transferring visible debris or glass beads into the purification tube.
Add 500 µl Buffer MCB, 30 µl MagPure RNA Particles and 20 µl Proteinase K. Mix up and down 20–30 times, incubate at room temperature for 10 min, mix several times, place on the magnetic rack for 1 min and remove the supernatant.
Buffer MCB must be supplemented with isopropanol before use. Resuspend the particles evenly before pipetting.
Add 600 µl Buffer MW1 and vortex for 20 sec to resuspend the particles. Place on the magnetic rack for 1 min, remove the supernatant, briefly spin down residual liquid, remove it carefully and air-dry for 2 min.
This step reduces carryover before DNase digestion. Do not disturb the bead pellet during residual-liquid removal.
Add 300 µl DNase mixture, prepared from 290 µl DNase Buffer and 10 µl DNase I. Gently shake or vortex to resuspend the particles and incubate at room temperature for 15 min.
DNase contact depends on complete bead resuspension; a compact bead pellet gives uneven digestion.
Add 450 µl Buffer MCB and vortex for 20 sec. Incubate at room temperature for 5 min, mix up and down 2–3 times, place on the magnetic rack for 1 min and remove the supernatant.
This step restores binding conditions after DNase treatment before the final wash sequence.
Add 600 µl Buffer MW1, vortex for 10 sec to resuspend the particles, place on the magnetic rack for 1 min and remove the supernatant.
Complete resuspension improves removal of salts, proteins and digestion carryover.
Add 600 µl Buffer RW2, vortex for 10 sec to resuspend the particles, place on the magnetic rack for 1 min and remove the supernatant.
Buffer RW2 must be supplemented with ethanol before use.
Repeat the RW2 wash once using 600 µl Buffer RW2, then magnetically separate and remove the wash liquid.
The second RW2 wash improves removal of residual salts and alcohol-soluble impurities.
Briefly spin the tube to collect liquid on the wall and cap, place it back on the magnetic rack, remove all residual liquid carefully and dry at room temperature or 37°C for 10 min.
Residual ethanol may inhibit downstream reactions; excessive drying can make the particles difficult to resuspend.
Add 30–100 µl RNase-free Water, vortex to resuspend the particles completely and incubate at room temperature for 3 min.
Use a smaller volume for higher concentration, or a larger volume when recovery is the priority.
Place the tube on the magnetic rack for 3 min. Transfer the supernatant containing purified RNA to a new RNase-free 1.5 ml tube and store at -80°C.
Do not carry magnetic particles into the final RNA eluate.
Collect about 1 × 10⁸ bacterial cells. Add the specified glass beads and 600 µl Buffer RTL, vortex at maximum speed for 10 min and centrifuge at 10,000 × g for 3 min at room temperature.
Cell wall disruption and complete clarification are the main control points for bacterial samples. Do not transfer bead debris or dense sediment into binding.
Transfer 500 µl lysate or clarified supernatant to a clean 1.5 ml tube.
For tissue, plant, yeast and bacterial samples, avoid transferring visible debris or glass beads into the purification tube.
Add 500 µl Buffer MCB, 30 µl MagPure RNA Particles and 20 µl Proteinase K. Mix up and down 20–30 times, incubate at room temperature for 10 min, mix several times, place on the magnetic rack for 1 min and remove the supernatant.
Buffer MCB must be supplemented with isopropanol before use. Resuspend the particles evenly before pipetting.
Add 600 µl Buffer MW1 and vortex for 20 sec to resuspend the particles. Place on the magnetic rack for 1 min, remove the supernatant, briefly spin down residual liquid, remove it carefully and air-dry for 2 min.
This step reduces carryover before DNase digestion. Do not disturb the bead pellet during residual-liquid removal.
Add 300 µl DNase mixture, prepared from 290 µl DNase Buffer and 10 µl DNase I. Gently shake or vortex to resuspend the particles and incubate at room temperature for 15 min.
DNase contact depends on complete bead resuspension; a compact bead pellet gives uneven digestion.
Add 450 µl Buffer MCB and vortex for 20 sec. Incubate at room temperature for 5 min, mix up and down 2–3 times, place on the magnetic rack for 1 min and remove the supernatant.
This step restores binding conditions after DNase treatment before the final wash sequence.
Add 600 µl Buffer MW1, vortex for 10 sec to resuspend the particles, place on the magnetic rack for 1 min and remove the supernatant.
Complete resuspension improves removal of salts, proteins and digestion carryover.
Add 600 µl Buffer RW2, vortex for 10 sec to resuspend the particles, place on the magnetic rack for 1 min and remove the supernatant.
Buffer RW2 must be supplemented with ethanol before use.
Repeat the RW2 wash once using 600 µl Buffer RW2, then magnetically separate and remove the wash liquid.
The second RW2 wash improves removal of residual salts and alcohol-soluble impurities.
Briefly spin the tube to collect liquid on the wall and cap, place it back on the magnetic rack, remove all residual liquid carefully and dry at room temperature or 37°C for 10 min.
Residual ethanol may inhibit downstream reactions; excessive drying can make the particles difficult to resuspend.
Add 30–100 µl RNase-free Water, vortex to resuspend the particles completely and incubate at room temperature for 3 min.
Use a smaller volume for higher concentration, or a larger volume when recovery is the priority.
Place the tube on the magnetic rack for 3 min. Transfer the supernatant containing purified RNA to a new RNase-free 1.5 ml tube and store at -80°C.
Do not carry magnetic particles into the final RNA eluate.
This workflow separates sample-specific Buffer RTL lysis / disruption from the shared magnetic silica-particle purification route. It is intended as a practical companion to the product manual rather than a replacement for the official protocol. Animal tissue and plant tissue enter after homogenization and clarification; cultured cells enter after direct lysis and lysate shearing; yeast and bacterial samples enter after glass-bead disruption and clarification. The downstream route follows MCB-adjusted RNA binding with MagPure RNA Particles and Proteinase K, MW1 wash, on-bead DNase digestion, post-DNase MCB rebinding, MW1 / RW2 washing, controlled drying and RNase-free water elution.
| Sample route | Displayed preparation estimate | Typical manual workflow time |
|---|---|---|
| Animal Tissue | 10 min | 75–95 min |
| Plant Tissue | 14 min | 80–105 min |
| Cultured Cells | 8 min | 72–90 min |
| Yeast Cells | 17 min | 85–105 min |
| Bacterial Cells | 17 min | 85–105 min |
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, short spin, 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 R6622 workflow, the main timing variables are sample disruption and clarification, lysate viscosity, bead resuspension, residual-liquid removal and final drying.
R6622 uses Buffer RTL lysis followed by MCB-adjusted magnetic silica-particle binding. Proteinase K is included during the primary binding stage, DNase digestion is performed on bead-bound RNA, and MCB is added again after DNase treatment to re-establish binding conditions before final washing.
The main control points are sample input, clarification and bead handling. Keep within the stated input limits, avoid transferring debris or glass beads, fully resuspend the particles during binding, DNase digestion and elution, and remove residual wash liquid without disturbing the bead pellet. After RW2 washing, dry enough to remove ethanol but avoid over-drying.