Tissue/Cell RNA/DNA Extraction Kit(SpinColumn)
Packing Specification：
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R2105-20
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Tissue/Cell RNA/DNA Extraction Kit(SpinColumn)
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20T
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CNY935
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R2105-50
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Tissue/Cell RNA/DNA Extraction Kit(SpinColumn)
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50T
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CNY1680
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R2105-200
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Tissue/Cell RNA/DNA Extraction Kit(SpinColumn)
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200T
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CNY6050
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For research use only. Not for use in medicine, clinical diagnosis, food, cosmetics or other applications.
Product Introduction：
This kit is designed for the rapid simultaneous extraction and separation of genomic DNA (gDNA) and total RNA from the same animal cell or tissue sample. The unique lysis buffer rapidly lyses cells and inactivates cellular RNA/DNA enzymes. The lysis mixture is then passed through a genomic DNA spin column, where gDNA is adsorbed while RNA passes through the filter. The gDNA adsorbed on the DNA spin column is eluted to obtain pure gDNA after a series of washing-centrifugation steps. After adjusting the binding conditions of the filtered RNA with ethanol, the RNA selectively binds to the RNA spin column under high chaotropic salt conditions. Pure RNA is then obtained through a series of rapid washing-centrifugation-elution steps. Based on the rapid phenol- and chloroform-free DNA/RNA extraction technology combined with the company's exclusive separation technology, the obtained RNA and gDNA are of high purity and do not interfere with each other. The RNA does not require DNase digestion and can be directly used in experiments such as reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qPCR). The gDNA can also be directly used in various downstream experiments such as Southern blotting, restriction enzyme digestion, and PCR.
Product Features：
1.No toxic reagents such as phenol or chloroform are used, and ethanol precipitation steps are eliminated.
2.Fast and simple operation; the simultaneous extraction and separation of RNA/gDNA from a single sample can generally be completed within 40 minutes.
3.The kit's exclusive spin columns and formulations ensure effective removal of gDNA residues. Under normal circumstances, the obtained RNA does not require DNase digestion and can be directly used in experiments such as RT-PCR and qPCR.
4.Multiple column washing steps ensure high purity of RNA/gDNA, which can be directly used in various downstream experiments.
Operation Steps：
Note: Before first use, add the specified volume of anhydrous ethanol to the Washing Buffer RW and Washing Buffer WB bottles, mix thoroughly, and mark the bottles with a marker. Add β-mercaptoethanol to RLT Plus to a final concentration of 2%. For example: add 12 μL β-mercaptoethanol to 600 μL RLT Plus.
Pre-Experiment Preparation
1.Preheat the incubator or water bath to 55℃ in advance.
2.Prepare DNase I digestion reaction solution in advance. The system can be configured according to the number of samples:
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Buffer
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Volume per Prep
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10 Preps
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DNase I Buffer
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45 μL
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450 μL
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RNase-free DNase I
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5 μL
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50 μL
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Total volume
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50 μL
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500 μL
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Note: DNase I is very sensitive to physical denaturation. Do not vortex vigorously to mix; invert gently to mix well.
Experimental Steps
1.Cultured Cells
a.Collect <107 suspension cells into a 1.5 mL centrifuge tube. For adherent cells, lyse directly in the culture plate; for cells cultured in flasks, digest with trypsin first, then pipette to collect.
b.Centrifuge at 13,000 rpm for 10 seconds (or 300×g for 5 minutes) to pellet the cells. Completely aspirate and discard the supernatant, leaving the cell pellet. Note that incomplete removal of the supernatant will dilute the lysis buffer, leading to reduced yield and purity.
c.Flick the tube wall to fully resuspend the cell pellet. Add 350 μL (<5×106 cells) or 600 μL (5×106-1×107 cells) of Lysis Buffer RLT Plus (ensure β-mercaptoethanol has been added to a final concentration of 2%), pipette to mix, then vigorously vortex by hand for 20 seconds to fully lyse the cells.
d.Homogenization: (Not required for very small cell amounts <1×105; vortex for 1 minute to homogenize). Vigorously pipette the lysate 5-10 times with a disposable 1 mL syringe fitted with a blunt 0.9 mm needle (or homogenize electrically for 30 seconds) to shear DNA, reduce viscosity, prevent column clogging, and improve yield.
e.Load the entire lysis mixture or homogenized mixture onto a DNA Spin Column (place the column in a collection tube).
f.Proceed directly to Step 3 in the "General Initial Steps" below.
2.Animal Tissues (e.g., Mouse Liver, Brain)
a.Electrical Homogenization: Quickly cut fresh tissue into small pieces with a scalpel. Add 350 μL (<15 mg tissue) or 600 μL (15-30 mg tissue) of Lysis Buffer RLT Plus (ensure β-mercaptoethanol has been added to a final concentration of 2%), then homogenize thoroughly electrically for 20-40 seconds.
b.Liquid Nitrogen Grinding + Homogenization: Grind the tissue into fine powder in liquid nitrogen. Transfer an appropriate amount of tissue powder (15 mg/30 mg) into a 1.5 mL centrifuge tube containing 350 μL/600 μL of Lysis Buffer RLT Plus (ensure β-mercaptoethanol has been added to a final concentration of 2%). Vigorously vortex by hand for 20 seconds to fully lyse the tissue. Vigorously pipette the lysate 5-10 times with a disposable 1 mL syringe fitted with a blunt 0.9 mm needle (or homogenize electrically for 30 seconds) to shear DNA, reduce viscosity, prevent column clogging, and improve yield.
c.Centrifuge the homogenized lysate at 13,000 rpm for 3 minutes to pellet any difficult-to-lyse debris or insoluble matter.
d.Load the entire supernatant of the lysate onto a DNA Spin Column (place the column in a collection tube).
e.Proceed directly to Step 3 in the "General Initial Steps" below.
General Initial Steps
3.Immediately centrifuge at 13,000 rpm for 1 minute. Retain the filtrate in the collection tube (RNA is in the filtrate), and gDNA is adsorbed on the filter membrane of the purification column (gDNA is adsorbed on the membrane). The DNA can be stored at 4℃ for short-term use. Ensure all liquid has passed through the filter after centrifugation with no residues on the membrane. If necessary, increase the centrifugal force and time.
RNA Extraction Steps
4.Add 50 μL or 80 μL Proteinase K to the filtrate from Step 3 of the "General Initial Steps" and vortex to mix.
5.Add 200 μL or 350 μL anhydrous ethanol, mix thoroughly (flocculent precipitation may form at this step but will not affect RNA extraction), and incubate at room temperature for 10 minutes.
6.Immediately load the mixture (≤700 μL per load; load in two batches if the volume exceeds 700 μL) into a SpinRA RNA Spin Column (place the column in a collection tube). Centrifuge at 13,000 rpm for 30 seconds and discard the waste liquid.
Retain the collection tube of the DNA Spin Column containing the filtrate and ethanol mixture. Place the DNA Spin Column back into the collection tube and store at 4℃ for subsequent gDNA extraction.
7.Add 350 μL of Deproteinization Buffer RW1, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.
8.Add 50 μL of the pre-prepared DNase I digestion working solution to the center of the silica matrix membrane of the SpinRA RNA Spin Column, and incubate at room temperature for 15 minutes.
9.Add 350 μL of Deproteinization Buffer RW1, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.
10.Add 500 μL of Washing Buffer RW (please check if anhydrous ethanol has been added first!), centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.
11.Add another 500 μL of Washing Buffer RW, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.
12.Place the SpinRA RNA Spin Column back into an empty collection tube, centrifuge at 13,000 rpm for 2 minutes to thoroughly remove residual washing buffer, as ethanol residues in the washing buffer may inhibit downstream reactions.
13.Remove the SpinRA RNA Spin Column and place it into an RNase-free centrifuge tube. Add 30-50 μL of RNase-free water to the center of the adsorption membrane (preheating the water in a 70-90℃ water bath in advance can improve yield) based on the expected RNA yield. Incubate at room temperature for 1 minute, then centrifuge at 12,000 rpm for 1 minute.
Note: To obtain higher RNA concentration, reuse the first eluate by adding it back to the spin column and repeating Step 11. Alternatively, if the expected RNA yield is >30 μg, repeat Step 11 with 30-50 μL of RNase-free water and combine the two eluates. Eluting twice yields RNA with higher concentration. Combining eluates from two separate elutions results in 15-30% higher RNA yield but lower concentration. Choose the method based on experimental needs.
gDNA Extraction Steps
14.Add 250 μL of Inhibitor Removal Buffer IR to the DNA Spin Column from Step 3 of the "General Initial Steps", centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.
15.Add 20 μL Proteinase K to 60 μL Inhibitor Removal Buffer IR, pipette to mix, then add dropwise to the center of the membrane of the DNA Spin Column, and incubate at room temperature for 5 minutes.
16.Add 250 μL of Inhibitor Removal Buffer IR, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.
17.Add 500 μL of Washing Buffer WB (please check if anhydrous ethanol has been added first!), centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.
18.Add another 500 μL of Washing Buffer WB, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.
19.Place the DNA Spin Column back into an empty collection tube, centrifuge at 13,000 rpm for 2 minutes to thoroughly remove residual washing buffer, as ethanol residues in the washing buffer may inhibit downstream reactions.
20.Remove the DNA Spin Column and place it into a clean centrifuge tube. Add 100 μL of Elution Buffer EB to the center of the adsorption membrane (preheating the elution buffer in a 65-70℃ water bath in advance yields better results). Incubate at room temperature for 3-5 minutes, then centrifuge at 12,000 rpm for 1 minute. Load the obtained solution back into the spin column, incubate at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 1 minute.
Note: Larger elution volume results in higher elution efficiency. If higher DNA concentration is required, the elution volume can be appropriately reduced, but the minimum volume should not be less than 50 μL. Excessively small volume reduces DNA elution efficiency and yield. DNA can be stored at 2-8℃ for short-term use or at -20℃ for long-term storage.
