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Tissue/Cell RNA/DNA/Protein Extraction Kit(SpinColumn)

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Product Description
Tissue/Cell RNA/DNA/Protein Isolation KitRapid simultaneous isolation of DNA, RNA and Protein from the same animal cell or tissue sample. No organic reagents such as phenol or chloroform are required. Unique lysis buffer rapidly lyses cells and inactivates cellular RNases/DNases. When passing through the genomic DNA adsorption column, DNA is bound to the column while RNA and Protein flow through, followed by multiple column washing steps. DNA, RNA and Protein with high purity, no impurity contamination and no mutual interference can be isolated simultaneously within approximately 60 minutes.

The obtained RNA can be used in various molecular biology experiments such as RT-PCR, Real Time PCR, Northern blot, Dot blot, poly A selection, in vitro translation, and cDNA library construction. Genomic DNA can also be used in downstream experiments such as Southern blotting, enzyme digestion, PCR and other assays. Protein can be used in SDS-PAGE electrophoresis, western blot and other experiments.

Product Features:
No contamination: The entire kit is RNase-Free treated.

Specificity: Suitable for animal tissues and cultured cells; simultaneous isolation of total RNA and genomic DNA without mutual interference.

Easy to operate: Simple procedure; the entire extraction takes approximately 60 minutes.

Safety: No organic reagents such as phenol or chloroform required.

Adsorption column: Equipped with specific adsorption columns. Extraction is performed via wash-centrifugation-elution steps.

High quality: Effectively ensures integrity of RNA/DNA/Protein with high recovery efficiency and high purity, free of protein and other impurities. Suitable for various molecular biology experiments.

Protocol: (Please read notes before the experiment)
Pre-warming Elution Buffer EB in a water bath at 65°C–70°C will give better results.

Sample Preparation:
1.Animal tissue: Mince fresh or -80°C frozen animal tissue as finely as possible. Add 350 μl (<20 mg tissue) or 600 μl (20–30 mg tissue) Lysis Buffer RLT and homogenize. Alternatively, grind into fine powder in liquid nitrogen, transfer 20 mg/30 mg powder into a 1.5 ml centrifuge tube containing 350 μl/600 μl Lysis Buffer RLT, mix vigorously by vortexing or pipetting with a 200 μl tip.
2.Adherent cells: Directly add Lysis Buffer RLT to the culture plate at 1 ml per 10 cm², mix by pipetting. Pipette repeatedly until no obvious cell clumps are visible. Insufficient lysis buffer may cause DNA contamination.
3.Suspension cells: Harvest cells by centrifugation. Add 350 μl Lysis Buffer RLT (<5×10⁶ cells) or 600 μl Lysis Buffer RLT (5×10⁶ – 1×10⁷ cells). Pipette repeatedly until no obvious cell clumps are visible. No washing is required to avoid mRNA degradation.

Extraction Procedure:
1.Incubate the treated sample at room temperature for 5–10 minutes, centrifuge at 13,000 rpm for 3 minutes; impurities will form a pellet.
2.Transfer the supernatant to a centrifuge tube containing Genomic DNA Adsorption Column DA. (Do not aspirate the pellet or floating debris. Take care not to introduce floating particles adhering to the outside of the pipette tip into the tube.)
3.Centrifuge at 13,000 rpm for 60 seconds. Keep the flow-through (contains RNA and Protein) and Column DA (contains DNA).(The experiment splits here. RNA extraction is recommended first. Column DA can be stored at 4°C temporarily. Protein is isolated from the flow-through in RNA Step 5.)

RNA Isolation Steps:
4.Estimate the volume of the flow-through, add an equal volume of 70% ethanol (please check that absolute ethanol has been added previously), mix immediately by pipetting.
5.Apply the mixture (<700 μl per load) to Adsorption Column RA in a collection tube. Centrifuge at 13,000 rpm for 30 seconds. (Save the flow-through for Protein extraction.)
6.Place Column RA into a new collection tube, add 700 μl Protein Removal Buffer RW1, incubate at room temperature for 1 minute, centrifuge at 12,000 rpm for 30 seconds, discard the waste.
7.Add 500 μl Wash Buffer RW (please check that absolute ethanol has been added previously), centrifuge at 12,000 rpm for 30 seconds, discard the waste.
8.Repeat Step 7 once.
9.Place Column RA back into the collection tube, centrifuge at 13,000 rpm for 2 minutes to remove residual wash buffer as much as possible to avoid inhibition of downstream reactions by residual ethanol; discard the waste tube.
10.Remove Column RA (avoid contact between the bottom of Column RA and waste in the collection tube), place into a new RNase-free centrifuge tube, add 30–50 μl RNase-free H₂O to the center of the adsorption membrane, incubate at room temperature for 1 minute, centrifuge at 12,000 rpm for 1 minute. Eluted RNA can be used immediately or stored at -80°C to prevent degradation.

DNA Isolation Steps:
11.Add 500 μl Inhibitor Removal Buffer IR to Column DA from Step 3, centrifuge at 12,000 rpm for 30 seconds, discard the waste.
12.Add 700 μl Wash Buffer WB (please check that absolute ethanol has been added previously), centrifuge at 12,000 rpm for 30 seconds, discard the waste.
13.Add 500 μl Wash Buffer WB, centrifuge at 12,000 rpm for 30 seconds, discard the waste.
14.Place DNA Adsorption Column DA into the collection tube, centrifuge at 13,000 rpm for 2 minutes to remove residual wash buffer as much as possible to avoid inhibition of downstream reactions by residual ethanol.
15.Remove Column DA (avoid contact between the bottom of Column DA and waste in the collection tube), place into a clean centrifuge tube, add 100 μl Elution Buffer EB to the center of the adsorption membrane, incubate at room temperature for 3–5 minutes, centrifuge at 12,000 rpm for 1 minute. Reapply the eluate to the same column, incubate at room temperature for 2 minutes, centrifuge at 12,000 rpm for 1 minute. Eluted DNA can be used immediately or stored at -20°C to prevent degradation.

Protein Isolation Steps:
16.Add an equal volume of Buffer APP to the flow-through from Step 5, vortex to mix thoroughly, incubate at room temperature for 15 minutes to precipitate Protein.
17.Centrifuge at 13,000 rpm for 5–10 minutes, carefully discard the supernatant. Add 500 μl 70% ethanol (please check that absolute ethanol has been added previously), invert to mix and centrifuge for 1 minute, retain the protein pellet. Remove residual supernatant completely with a pipette.
18.Air-dry the pellet at room temperature for 5–10 minutes to evaporate ethanol completely to avoid affecting downstream experiments.
19.Dissolve the protein pellet in 30–150 μl 1× Protein Loading Buffer or other buffer required for downstream experiments.
Incubate at 95°C for 5 minutes to dissolve and denature the protein, cool to room temperature, centrifuge at maximum speed for 1 minute.