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S.aureus RNA Extraction Kit(SpinColumn)

Packing Specification：

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 RT-PCR, quantitative real-time PCR, and other experiments. The unique lysis buffer rapidly lyses 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 RT-PCR, quantitative real-time PCR, and other experiments.
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：
Note: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!
1.Add all 500 μL of Resuscitation Buffer to the Staphylococcal Lysozyme, invert to mix well and dissolve.

2.Scrape an appropriate amount of bacteria (about the size of half a mung bean), suspend in 90 μL of RNase-free H₂O, pipette to mix well. Then add 10 μL of the Staphylococcal Lysozyme solution and pipette to mix well.

3.Incubate at room temperature for 5-10 minutes to ensure complete lysis.

4.Add 500 μL of Lysis Buffer RLT Plus, pipette to mix well, then vortex vigorously for 20 seconds to fully lyse.
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. Take the lysate supernatant for the next step.

5.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.

6.Use a micropipette to accurately estimate the volume of the filtrate (usually 600 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.

7.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.

8.Add 700 μL of Deproteinization Buffer RW1, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

9.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.

10.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.

11.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.