Bacteria RNA Extraction Kit with gDNA Eraser(SpinColumn)
Packing Specification：
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R2120-20
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Bacteria RNA Extraction Kit with gDNA Eraser(SpinColumn)
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20T
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CNY750
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R2120-50
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Bacteria RNA Extraction Kit with gDNA Eraser(SpinColumn)
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50T
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CNY1360
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R2120-200
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Bacteria RNA Extraction Kit with gDNA Eraser(SpinColumn)
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200T
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CNY4900
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For research use only. Not for use in medicine, clinical diagnosis, food, cosmetics or other applications.
Product Introduction:
Based on our rapid RNA extraction technology without phenol or chloroform, we have successfully developed genomic DNA removal column technology to ensure effective elimination of gDNA residue. The obtained RNA does not require DNase digestion and can be directly used in PCR, quantitative real-time PCR, and other experiments. The unique lysis buffer rapidly lyses bacterial cells and inactivates cellular RNases. The lysis mixture is then passed through a genomic DNA removal column, where genomic DNA is retained while RNA passes through. After adjusting the binding conditions of the filtered RNA with ethanol, RNA is selectively adsorbed to the silica matrix membrane in the spin column under high chaotropic salt conditions. Through a series of rapid washing and centrifugation steps, deproteinization buffer and wash buffer remove impurities such as cellular metabolites and proteins. Finally, pure RNA is eluted from the silica matrix membrane with low-salt RNase-free H2O.
Product Features:
1.The silica matrix membrane in the spin column is made of high-quality special adsorption membrane, with minimal differences in adsorption capacity between columns.
2.No toxic reagents such as phenol or chloroform are used, and no ethanol precipitation steps are required.
3.Fast and simple: Operation for a single sample can generally be completed within 30 minutes.
4.Genomic DNA removal column technology ensures effective elimination of gDNA residue. The obtained RNA does not require DNase digestion and can be directly used in PCR, quantitative real-time PCR, and other experiments.
5.Multiple column washing steps ensure high purity: The typical OD260/OD280 ratio ranges from 2.1 to 2.2 (The ratio of 100% pure RNA is generally around 2.2).
Experimental Procedures:
Notes
Before first use, add the indicated amount of absolute ethanol to the Wash Buffer RW bottle and 70% Ethanol bottle, mix thoroughly, and mark with a check in the box immediately after adding to avoid repeated addition!
To extract bacterial RNA, first prepare TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) supplemented with lysozyme to a final concentration of 1 mg/mL.
1.Centrifuge 1-2 mL of bacterial culture (approximately 10⁸-10⁹ cells) into a 1.5 mL centrifuge tube. Remove the supernatant as much as possible, ensuring the residual supernatant does not exceed 20 μL per 100 μL of TE buffer used.
2.Resuspend the cells thoroughly in 100 μL (for <5×10⁸ cells) or 200 μL (for 5×10⁸-7.5×10⁸ cells) of TE buffer (10 mM Tris-HCl, 1 mM EDTA). The TE buffer should be pre-supplemented with lysozyme or lysostaphin to a final concentration of 1 mg/mL. Alternatively, resuspend the cells in TE buffer first, then add a small amount of lysozyme with a clean pipette tip.
3.Incubate at room temperature (15-25°C) for 5 minutes (for lysozyme) or at 37°C for 15 minutes (for lysostaphin) to lyse the cell wall. Vortex for 10 seconds every 2 minutes to assist in cell wall lysis. Note: The difficulty of cell wall lysis varies among bacterial species. Generally, the above conditions are sufficient for Gram-negative bacteria such as E. coli, and this step may even be omitted. However, some Gram-positive bacteria such as B. subtilis require a higher lysozyme concentration (15 mg/mL) and longer incubation time (10 minutes). For Staphylococcus aureus, add lysostaphin to 1 mg/mL and incubate at 37°C for 15 minutes. In summary, adjust the enzyme type, working concentration, incubation temperature, and time according to specific bacterial strains. For bacteria with tough cell walls, additional methods such as glass bead beating, mechanical lysis, or Proteinase K digestion can be combined to assist in lysis.
4.Centrifuge briefly for 1 minute to pellet the cells at the bottom of the tube, then aspirate and discard the supernatant. Vortex to resuspend and disperse the cells.
5.Add 500 μL of Lysis Buffer RLT Plus, pipette to mix well, then vortex vigorously by hand for 20 seconds to fully lyse the cells. Generally, no obvious clumps or insoluble substances should be visible after adding the lysis buffer and thorough vortexing. In rare cases where clumps or insoluble substances remain, centrifuge the lysate at 13,000 rpm for 3 minutes to pellet undigested debris or insoluble material. Transfer the supernatant to proceed to the next step.
6.Immediately add the lysate to a genomic DNA removal column (place the column into a collection tube), centrifuge at 13,000 rpm for 60 seconds, and retain the filtrate (RNA is in the filtrate). Ensure that all liquid has passed through the membrane after centrifugation with no residue left on the membrane. If necessary, increase the centrifugal force and time.
7.Use a micropipette to accurately estimate the volume of the filtrate (usually 500 μL; subtract the volume lost during filtration), add an equal volume of 70% ethanol (please check if absolute ethanol has been added first!). Precipitation may occur at this time, but it will not affect the extraction process. Immediately pipette to mix well; do not centrifuge.
8.Immediately transfer the mixed solution (less than 700 μL each time, can be added in two portions) to a SpinRA column (place the spin column into a collection tube), centrifuge at 13,000 rpm for 60 seconds, and discard the waste liquid.
9.Add 700 μL of Deproteinization Buffer RW1, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.
10.Add 500 μL of Wash Buffer RW (please check if absolute ethanol has been added first!), centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid. Repeat this step with another 500 μL of Wash Buffer RW.
11.Place the SpinRA column back into the empty collection tube, centrifuge at 13,000 rpm for 2 minutes to remove as much wash buffer as possible, avoiding residual ethanol in the wash buffer that may inhibit downstream reactions.
12.Remove the SpinRA column, place it into a new RNase-free centrifuge tube. According to the expected RNA yield, add 30-50 μL of RNase-free water to the center of the adsorption membrane (heating the water in a 70-90°C water bath in advance can improve yield), incubate at room temperature for 1 minute, and centrifuge at 12,000 rpm for 1 minute.
If the expected RNA yield is >30 μg, add 30-50 μL of RNase-free water and repeat Step 12, then combine the two eluates. Alternatively, add the first eluate back to the spin column and repeat Step 12 for higher RNA concentration. Repeating the elution once results in a high-concentration RNA eluate. Combining two separate eluates increases the RNA yield by 15–30% compared to a single elution, but the concentration is lower. Users can choose according to their needs.
