Cat. No. R413002 / R413003
Sample-specific MagZol lysis, chloroform phase separation and silica-column total RNA purification.
Homogenize 30–100 mg tissue in 1 ml MagZol Reagent using a mechanical homogenizer, or pulverize the sample in liquid nitrogen and transfer the powder into a clean tube with MagZol.
The sample volume should not exceed 10% of the MagZol volume. For plant, fungal or yeast-rich material, mechanical or enzymatic homogenization may be required before phase separation.
For samples rich in fat, protein, polysaccharides or extracellular material, centrifuge the homogenate at 12,000 × g for 10 min at 4°C and transfer the supernatant.
Optional steps are not included in the standard cumulative timeline unless selected for the sample.
Add 0.2 ml chloroform per 1 ml MagZol Reagent. Cap securely, shake vigorously by hand for 15 s and incubate at room temperature for 3 min.
This chloroform mixing and 3 min incubation step is marked as critical in the manual and should not be modified.
Centrifuge at 12,000 × g for 15 min at 4°C. Transfer only the upper aqueous phase to a new tube without disturbing the interphase or organic phase.
RNA remains in the aqueous phase after separation.
Add 1 volume, usually about 550 µl, of Buffer RW2 to the aqueous phase and mix thoroughly by vortexing. Do not centrifuge.
Visible precipitates after ethanol-containing buffer addition should be fully resuspended before loading.
Insert a HiPure RNA Mini Column into a 2 ml collection tube. Load 600 µl of the mixture and centrifuge at 10,000 × g for 1 min; repeat until all sample has passed through the column.
Repeated loading is included because the adjusted aqueous phase may exceed one column load.
Add 650 µl Buffer RW1 and centrifuge at 10,000 × g for 1 min. Discard the filtrate and reuse the collection tube.
Optional steps are not included in the standard cumulative timeline unless selected for the sample.
Add 650 µl Buffer RW2 and centrifuge at 10,000 × g for 1 min. Discard the filtrate and reuse the collection tube.
Buffer RW2 must be prepared with absolute ethanol before use.
Centrifuge the empty column at 10,000 × g for 2 min to dry the column matrix.
Complete removal of residual ethanol is important for downstream RT-PCR performance.
Transfer the column to a clean 1.5 ml tube. Add 30–50 µl RNase Free Water to the membrane center, let sit for 2 min and centrifuge at 10,000 × g for 1 min.
This is the first elution from the silica membrane.
Repeat the elution step using another volume of RNase Free Water, or reload the first eluate onto the membrane when a higher RNA concentration is required.
The manual lists this as the next workflow step; fresh water supports higher recovery, while re-elution supports concentration-sensitive workflows.
Store purified RNA at -20°C, or use an appropriate lower-temperature condition for longer storage.
Avoid repeated freeze-thaw cycles where possible.
Pellet suspension cells by centrifugation and lyse by repetitive pipetting in MagZol Reagent. Use 1 ml MagZol per 5 × 10⁶ animal, plant or yeast cells, or per 1 × 10⁸ bacterial cells.
Avoid washing cells before adding MagZol, as this can increase mRNA degradation and RNase contamination risk. Plant, fungal and yeast cells may require additional mechanical or enzymatic homogenization.
Add 0.2 ml chloroform per 1 ml MagZol Reagent. Cap securely, shake vigorously by hand for 15 s and incubate at room temperature for 3 min.
This chloroform mixing and 3 min incubation step is marked as critical in the manual and should not be modified.
Centrifuge at 12,000 × g for 15 min at 4°C. Transfer only the upper aqueous phase to a new tube without disturbing the interphase or organic phase.
RNA remains in the aqueous phase after separation.
Add 1 volume, usually about 550 µl, of Buffer RW2 to the aqueous phase and mix thoroughly by vortexing. Do not centrifuge.
Visible precipitates after ethanol-containing buffer addition should be fully resuspended before loading.
Insert a HiPure RNA Mini Column into a 2 ml collection tube. Load 600 µl of the mixture and centrifuge at 10,000 × g for 1 min; repeat until all sample has passed through the column.
Repeated loading is included because the adjusted aqueous phase may exceed one column load.
Add 650 µl Buffer RW1 and centrifuge at 10,000 × g for 1 min. Discard the filtrate and reuse the collection tube.
Optional steps are not included in the standard cumulative timeline unless selected for the sample.
Add 650 µl Buffer RW2 and centrifuge at 10,000 × g for 1 min. Discard the filtrate and reuse the collection tube.
Buffer RW2 must be prepared with absolute ethanol before use.
Centrifuge the empty column at 10,000 × g for 2 min to dry the column matrix.
Complete removal of residual ethanol is important for downstream RT-PCR performance.
Transfer the column to a clean 1.5 ml tube. Add 30–50 µl RNase Free Water to the membrane center, let sit for 2 min and centrifuge at 10,000 × g for 1 min.
This is the first elution from the silica membrane.
Repeat the elution step using another volume of RNase Free Water, or reload the first eluate onto the membrane when a higher RNA concentration is required.
The manual lists this as the next workflow step; fresh water supports higher recovery, while re-elution supports concentration-sensitive workflows.
Store purified RNA at -20°C, or use an appropriate lower-temperature condition for longer storage.
Avoid repeated freeze-thaw cycles where possible.
Lyse cells directly in the culture dish with MagZol Reagent, using about 1 ml per 10 cm² dish area, and pass the lysate several times through a blue pipette tip.
Use more MagZol if the lysate is too viscous to aspirate; insufficient reagent can increase DNA contamination risk.
Add 0.2 ml chloroform per 1 ml MagZol Reagent. Cap securely, shake vigorously by hand for 15 s and incubate at room temperature for 3 min.
This chloroform mixing and 3 min incubation step is marked as critical in the manual and should not be modified.
Centrifuge at 12,000 × g for 15 min at 4°C. Transfer only the upper aqueous phase to a new tube without disturbing the interphase or organic phase.
RNA remains in the aqueous phase after separation.
Add 1 volume, usually about 550 µl, of Buffer RW2 to the aqueous phase and mix thoroughly by vortexing. Do not centrifuge.
Visible precipitates after ethanol-containing buffer addition should be fully resuspended before loading.
Insert a HiPure RNA Mini Column into a 2 ml collection tube. Load 600 µl of the mixture and centrifuge at 10,000 × g for 1 min; repeat until all sample has passed through the column.
Repeated loading is included because the adjusted aqueous phase may exceed one column load.
Add 650 µl Buffer RW1 and centrifuge at 10,000 × g for 1 min. Discard the filtrate and reuse the collection tube.
Optional steps are not included in the standard cumulative timeline unless selected for the sample.
Add 650 µl Buffer RW2 and centrifuge at 10,000 × g for 1 min. Discard the filtrate and reuse the collection tube.
Buffer RW2 must be prepared with absolute ethanol before use.
Centrifuge the empty column at 10,000 × g for 2 min to dry the column matrix.
Complete removal of residual ethanol is important for downstream RT-PCR performance.
Transfer the column to a clean 1.5 ml tube. Add 30–50 µl RNase Free Water to the membrane center, let sit for 2 min and centrifuge at 10,000 × g for 1 min.
This is the first elution from the silica membrane.
Repeat the elution step using another volume of RNase Free Water, or reload the first eluate onto the membrane when a higher RNA concentration is required.
The manual lists this as the next workflow step; fresh water supports higher recovery, while re-elution supports concentration-sensitive workflows.
Store purified RNA at -20°C, or use an appropriate lower-temperature condition for longer storage.
Avoid repeated freeze-thaw cycles where possible.
This workflow separates sample-specific MagZol lysis from the shared chloroform phase-separation and silica-column purification route. It is intended as a practical companion to the product manual rather than a replacement for the official protocol. The downstream steps follow a phenol / guanidine lysis, aqueous-phase recovery, ethanol-adjusted column-binding and wash-elution workflow.
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, aqueous-phase transfer, filtrate disposal and 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 the first sample-specific lysis step through the second elution / re-elution step to final storage.
R4130 is different from direct silica-column RNA kits because sample lysis is carried out in MagZol Reagent, a phenol / guanidine-based reagent used to disrupt samples and inhibit RNases. Chloroform phase separation is then used to separate RNA-containing aqueous phase from the organic phase and interphase before column binding. This design is useful for broad sample compatibility, but it requires careful handling of the phase boundary.
The most important handling point is clean recovery of the upper aqueous phase after centrifugation. Carryover from the interphase or organic phase may reduce RNA purity. Difficult matrices such as fatty, protein-rich, polysaccharide-rich, plant, fungal or yeast-containing samples may require additional clarification or stronger homogenization before the shared column workflow. The optional RW1 wash can be selected according to purity requirements. The second elution / re-elution is shown in the standard workflow because the manual lists it as a subsequent elution step; fresh RNase Free Water can be used for higher recovery, while reloading the first eluate can be used when higher RNA concentration is required.