This kit utilizes an optimized buffer system, specifically designed for extracting total RNA from bacterial samples. It can effectively isolate RNA molecules with a molecular weight greater than 200 nt.
The kit features high efficiency, rapid processing, and ease of use, eliminating the need for phenol-chloroform extraction steps. RNA purified with this kit exhibits high purity, with minimal contamination from proteins, genomic DNA, and other impurities. The isolated RNA can be directly used in various molecular biology experiments, including Northern blotting, dot blotting, mRNA purification, in vitro translation, RNase protection assays, RT-PCR, RT-qPCR, and cDNA library construction.
Storage Conditions
- R669890A, R669890B, R669890C, R669890D, R669890E, R669890F, R669890G, R669890H, R669890I: Stable for 15 months when stored at room temperature.
- R669890J, R669890K, R669890L: Stable for 15 months when stored at 2-8°C.
- Buffer URL supplemented with β-mercaptoethanol can be stored at 4°C for 1 month.
Precautions
1. Buffer URL Preparation: Before use, add β-mercaptoethanol to Buffer URL to a final concentration of 1%. The prepared Buffer URL can be stored at 4°C for 1 month. Precipitates may form in Buffer URL during storage; if precipitates occur, heat the solution at 37°C to dissolve, then allow it to cool to room temperature before use.
2. Sample Freshness: Use freshly harvested bacterial samples whenever possible. RNA yield depends on the integrity of RNA in the starting material; RNA degraded into small fragments during sample processing cannot be effectively recovered.
3. DNase I Stock Solution Preparation: Inject 550 μL of RNase-Free ddH₂O (from R669890L) into the vial containing DNase I powder using a 1 mL syringe, mix gently until dissolved. Aliquot the solution and store at -30 ~ -15°C (stable for 9 months). Prepare working DNase I solution as needed and store at 2-8°C (stable for 42 days). Avoid repeated freeze-thaw cycles.
Protocol
Preparatory Step: Add 48 mL of anhydrous ethanol to 12 mL of Buffer RW2 before use.
1. Harvest Bacterial Cells: Centrifuge the bacterial culture at 12,000 rpm (13,400 × g) for 2 min at 4°C to harvest cells (maximum cell number ≤ 1×10⁹). Carefully remove all supernatant. Subsequent centrifugation steps should be performed at room temperature.
2. Resuspend and Lyse Cells: Resuspend the cell pellet thoroughly in 100 μL of Buffer TE containing lysozyme. Incubate at 37°C. Refer to the table below for lysozyme concentration and incubation time:
Bacteria Type
| Final Lysozyme Concentration in Buffer TE
| Incubation Time at 37°C
|
Gram-negative (G⁻)
| 500 μg/mL
| 3-5 min
|
Gram-positive (G⁺)
| 3 mg/mL
| 5-10 min
|
TE Buffer Formula: 50 mM Tris (pH 8.0), 10 mM Na₂-EDTA (pH 8.0). Autoclave at 121°C for 20 min.
Example:
- For G⁻ bacteria: Mix 1 μL lysozyme stock solution (50 mg/mL) with 99 μL Buffer TE to achieve a final concentration of 500 μg/mL.
- For G⁺ bacteria: Mix 6 μL lysozyme stock solution (50 mg/mL) with 94 μL Buffer TE to achieve a final concentration of 3 mg/mL.
3. Lyse Cells: Add 350 μL of Buffer URL (supplemented with β-mercaptoethanol before use), vortex vigorously for 15 sec (precipitates may form). Transfer the entire mixture (including precipitates) to Filter Column GS (placed in a collection tube), centrifuge at 12,000 rpm (13,400 × g) for 1 min.
4. Bind RNA to Adsorption Column: Transfer the filtrate to a new 1.5 mL RNase-Free centrifuge tube (do not aspirate the bottom precipitate). Add 250 μL of anhydrous ethanol and mix well (precipitates may form). Transfer the entire mixture to Adsorption Column RA (placed in a collection tube), centrifuge at 12,000 rpm (13,400 × g) for 30 sec. Discard the flow-through and place the column back into the collection tube.
5. Wash Column: Add 350 μL of Buffer RW to the column, centrifuge at 12,000 rpm (13,400 × g) for 30 sec. Discard the flow-through and place the column back into the collection tube.
6. Prepare DNase I Working Solution: Combine 10 μL of DNase I stock solution with 70 μL of DNase I Buffer in a new RNase-Free centrifuge tube, mix gently.
7. Remove Genomic DNA: Add 80 μL of DNase I working solution to the center of the adsorption column membrane. Incubate at room temperature for 15 min.
8. Post-DNase Wash: Add 350 μL of Buffer RW to the column, centrifuge at 12,000 rpm (13,400 × g) for 30 sec. Discard the flow-through and place the column back into the collection tube.
9. Final Wash: Add 500 μL of Buffer RW2 (supplemented with anhydrous ethanol before use) to the column, centrifuge at 12,000 rpm (13,400 × g) for 30 sec. Discard the flow-through and place the column back into the collection tube.
10. Repeat Final Wash: Repeat step 9.
11. Dry Column: Centrifuge the column at 12,000 rpm (13,400 × g) for 2 min to remove residual ethanol.
12. Elute RNA: Transfer the adsorption column to a new 1.5 mL RNase-Free centrifuge tube. Open the column cap and air-dry for 1 min at room temperature to completely remove residual ethanol. Add 30-100 μL of RNase-Free ddH₂O dropwise to the center of the membrane, then centrifuge at 12,000 rpm (13,400 × g) for 30 sec. The eluted RNA can be used directly in downstream experiments or stored at ≤ -80°C.
Note: The volume of RNase-Free ddH₂O should not be less than 30 μL, as smaller volumes will reduce recovery. To increase RNA yield, re-apply the first eluate to the column for a second elution, or pre-heat the RNase-Free ddH₂O to 65°C before elution.
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