Bacteria RNA Extraction Kit(SpinColumn)
Packing Specification：
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R2119-20
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Bacteria RNA Extraction Kit(SpinColumn)
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20T
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CNY610
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R2119-50
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Bacteria RNA Extraction Kit(SpinColumn)
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50T
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CNY1100
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R2119-200
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Bacteria RNA Extraction Kit(SpinColumn)
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200T
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CNY3960
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For research use only. Not for use in medicine, clinical diagnosis, food, cosmetics or other applications.
Product Introduction:
The unique lysis buffer rapidly lyses bacterial cells and inactivates cellular RNases. After adjusting the binding conditions 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.Multiple column washing steps ensure high purity: The typical OD260/OD280 ratio ranges from 1.9 to 2.0, with almost no DNA residue. Suitable for RT-PCR, Northern blot, and various other experiments.
Experimental Procedures:
Notes
Before first use, add the indicated amount of absolute ethanol to the Wash Buffer RW 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) supplemented with lysozyme or lysostaphin to a final concentration of 1 mg/mL.
Experimental Procedures
1.Centrifuge 1-2 mL of bacterial culture (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 (Step 2).
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) supplemented with lysozyme or lysostaphin (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, and this step may even be omitted. However, some Gram-positive bacteria with tough cell walls require higher lysozyme concentrations, lysostaphin, mechanical lysis with glass beads, Proteinase K digestion, or a combination of methods. Adjust the enzyme concentration, incubation temperature, time, and select the appropriate method according to specific bacterial strains.
4.Add 350 μL of Lysis Buffer RLT (if 100 μL of TE/enzyme was used in Step 2) or 700 μL of Lysis Buffer RLT (if 200 μL of TE/enzyme was used). 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 entire supernatant to a new centrifuge tube and proceed to the next step.
5.Add 250 μL of 96-100% absolute ethanol (for tubes with 100 μL TE/350 μL RLT) or 500 μL of 96-100% absolute ethanol (for tubes with 200 μL TE/700 μL RLT). Immediately pipette to mix well.
6.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.
7.Add 700 μL of Deproteinization Buffer RW1, incubate at room temperature for 30 seconds, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid. If DNA residue is significant, incubate at room temperature for 5 minutes after adding RW1 before centrifugation.
8.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.
9.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.
10.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 10, then combine the two eluates. Alternatively, add the first eluate back to the spin column and repeat Step 10 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.
