Products

Allprep RNA/DNA/miRNA Extraction Kit(SpinColumn)

Packing Specification：

For research use only. Not for use in medicine, clinical diagnosis, food, cosmetics or other applications.

Product Introduction:
This kit uses a unique lysis buffer to rapidly lyse various samples such as animal tissues, plant tissues, and cultured cells. It solubilizes cellular contents, inhibits nuclease activity, and effectively prevents RNA degradation during extraction. Organic reagents are added for extraction to remove proteins and DNA. Under high-concentration ethanol conditions, RNA (including total RNA and small molecular RNAs such as microRNA, siRNA, snRNA, and other small RNAs <200 nt) binds to the special silica matrix membrane in the spin column. Through a series of rapid washing-centrifugation steps with special washing solutions, impurities such as cellular metabolites and proteins are further removed. Finally, pure RNA is eluted from the silica matrix membrane using a low-salt elution buffer.

The extracted RNA has high purity and yield, free from genomic DNA and protein contamination. It can be directly used for a series of operations including RT-PCR, Northern blot, dot blot, polyA selection, in vitro translation, RNase protection assay, and molecular cloning.

Product Features:
1.The silica matrix membranes in the spin columns are all high-quality specially manufactured adsorption membranes, ensuring minimal variation in adsorption capacity between columns.
2.Suitable for multiple sample types including animal tissues, plant tissues, and cultured cells.
3.Few steps and simple operation: processing of a single sample can generally be completed within 30 minutes.
4.Multiple column washing steps ensure high purity, with a typical OD260/OD280 ratio of 2.1-2.2 (the ratio of 100% pure RNA is generally around 2.2).

Application Scope:
Suitable for the rapid extraction and purification of total RNA and small molecular RNAs (microRNA, siRNA, snRNA, and other small RNAs <200 nt) from samples such as animal tissues, plant tissues, and cultured cells.

Operation Steps:
Note: Before first use, add the specified volume of absolute ethanol to Wash Solution 1 (BR1) and Wash Solution 2 (BR2), mix thoroughly. Mark the corresponding box immediately after adding ethanol to avoid repeated additions!

1.Sample Homogenization
1.1Animal Tissues
a.Liquid Nitrogen Grinding: Cut animal tissue into small pieces with surgical scissors, grind in liquid nitrogen. Quickly transfer 50~100 mg of ground tissue powder into a 2 mL centrifuge tube containing 1 mL of Lysis Buffer, and vortex vigorously for 20 seconds to lyse thoroughly.
b.Grinder Homogenization: Cut animal tissue into small pieces with surgical scissors. Quickly transfer 50~100 mg of cut tissue pieces into a 2 mL centrifuge tube containing 1 mL of Lysis Buffer (tissue volume should not exceed 10% of the Lysis Buffer volume). Homogenize using a low-temperature grinder.

1.2 Animal Cells
a.Adherent Cells:
Add 1 mL of Lysis Buffer directly to a 3.5 cm diameter culture dish to lyse cells, and mix gently by pipetting. The volume of Lysis Buffer required is determined by the culture dish area rather than the number of cells (1 mL per 10 cm²). For a standard-sized cell culture flask, add 1 mL of Lysis Buffer, shake immediately to ensure full contact between the buffer and all cells at the bottom to lyse cells and inactivate RNases, then mix gently by repeated pipetting. Insufficient Lysis Buffer may result in DNA contamination in the extracted RNA.
b.Suspension Cells:
Pellet cells by centrifugation and carefully discard the supernatant. Add 1 mL of Lysis Buffer per 5~10×106 animal cells, plant cells, or yeast cells, or per 1×107 bacterial cells. Lyse cells by repeated pipetting in the Lysis Buffer. Avoid washing cells before adding Lysis Buffer, as this may increase the risk of mRNA degradation.

1.3 Plant Tissues
a.Liquid Nitrogen Grinding: Cut plant tissue into small pieces with surgical scissors, grind in liquid nitrogen. Quickly transfer 60~100 mg of ground tissue powder into a 2 mL centrifuge tube containing 1 mL of Lysis Buffer, and vortex vigorously for 20 seconds to lyse thoroughly.
b.Grinder Homogenization: Cut plant tissue into small pieces with surgical scissors. Quickly transfer 60~100 mg of cut tissue pieces into a 2 mL centrifuge tube containing 1 mL of Lysis Buffer (tissue volume should not exceed 10% of the Lysis Buffer volume). Homogenize using a low-temperature grinder.

2.Incubate the sample-containing lysis buffer at 75℃ for 5 minutes, then cool to room temperature and let stand for 3-5 minutes.

3.Optional Step: Centrifuge at 12,000 rpm (~13,400 x g) at 4℃ for 5 minutes. Transfer the supernatant to a new user-prepared centrifuge tube (recommended for samples with high protein, fat, or polysaccharide content).

4.Add 0.2 mL of chloroform per 1 mL of Lysis Buffer. Tighten the tube cap, vortex vigorously for 15 seconds, and incubate at room temperature for 3 minutes.

5.Centrifuge at 12,000 rpm at 4℃ for 15 minutes using a high-speed refrigerated benchtop centrifuge. The sample will separate into three layers: the lower organic phase, the middle layer, and the upper colorless aqueous phase (containing RNA). The volume of the aqueous phase is approximately 50% of the added Lysis Buffer volume.

6.Carefully transfer the aqueous phase to a new tube (avoid touching the middle layer). Record the volume of the aqueous phase accurately. Add 1.5 volumes of absolute ethanol (must be at room temperature) and mix immediately by pipetting or inverting the tube several times. Precipitation may form after adding ethanol, but this will not affect the experiment. Do not centrifuge.

7.Load the mixture into Spin Columns AC (placed in collection tubes) in batches (≤700 μL per aliquot). Centrifuge at 12,000 rpm for 30-60 seconds. Discard the filtrate in the collection tube and place the adsorption column back into the collection tube.

8.Add 700 μL of Wash Solution 1 (check if absolute ethanol has been added!). Centrifuge at 12,000 rpm for 30 seconds. Discard the filtrate and place the adsorption column back into the collection tube.

9.Add 500 μL of Wash Solution 2 (check if absolute ethanol has been added!). Centrifuge at 12,000 rpm for 30 seconds. Discard the filtrate and place the adsorption column back into the collection tube.

10.Repeat Step 9: Add another 500 μL of Wash Solution 2 (check if absolute ethanol has been added!). Centrifuge at 12,000 rpm for 30 seconds. Discard the filtrate and place the adsorption column back into the collection tube.

11.Place the RNA adsorption column back into the empty collection tube. Centrifuge at 13,000 rpm for 2-5 minutes to completely remove residual washing solution (residual ethanol in the washing solution may inhibit downstream reactions).
Note: This step is critical to remove residual ethanol from the adsorption column RS. Ethanol residue can affect subsequent enzymatic reactions (e.g., restriction digestion, PCR).

12.Transfer the RNA adsorption column to a new user-prepared RNase-free centrifuge tube. Add 50-80 μL of RNase-free H2O (heating in a 70-90℃ water bath can improve yield) to the center of the adsorption membrane according to the expected RNA yield. Incubate at room temperature for 1-2 minutes, then centrifuge at 13,000 rpm for 1 minute to collect the RNA solution.Use the extracted RNA for reverse transcription immediately. Aliquot the remaining RNA solution and store at -80℃ to avoid repeated freeze-thaw cycles.
Remarks:For higher RNA concentration: Reapply the first eluate to the adsorption column and repeat Step 12.For expected RNA yield >10 μg: Add another 10-30 μL of RNase-free water and repeat Step 12, then combine the two eluates.Eluting twice yields higher concentration RNA. Combining eluates from two separate elutions increases RNA yield by 15-30% compared to a single elution but results in lower concentration. Choose the method based on experimental needs.Higher elution volume leads to higher elution efficiency. To obtain higher RNA concentration, the elution volume can be appropriately reduced, but the minimum volume should not be less than 30 μL (excessively small volume reduces RNA elution efficiency and yield).