Agarose Gel DNA Extraction Kit Spin Column Molecular Biology Life Science Research

Product Introduction

In the presence of a high-concentration chaotropic salt, DNA fragments selectively bind to the silica-based membrane in the spin column. Through a series of rapid washing and centrifugation steps, impurities such as primers, nucleotides, proteins, and enzymes are removed by the wash buffer. Finally, a low-salt, high-pH elution buffer is used to elute the purified DNA from the silica membrane.

I. Product Information：

II. Storage Condition：

1. This kit can be stored for 12 months under dry conditions at room temperature (15°C–25°C).

2. All solutions should be clear. If precipitation occurs due to low temperature, the solutions should not be used directly. Warming in a 37°C water bath for several minutes will restore clarity. Before use, allow the solutions to return to room temperature.

3. Storage at low temperatures (4°C or –20°C) may cause precipitation and affect performance; therefore, transport and storage should be carried out at room temperature (15°C–25°C).

4. To prevent evaporation, oxidation, or pH changes due to prolonged exposure to air, tightly cap all solution containers immediately after use.

III. Materials to Be Prepared by the User：

Absolute ethanol, benchtop high-speed centrifuge, constant-temperature water bath, pipettes, electrophoresis tank, gel imaging system, etc.; DNA Marker, loading dye, agarose, electrophoresis buffer (1× TAE or 0.5× TBE), 1.5 mL centrifuge tubes, etc.

IV. Product Description:

In the presence of a high-concentration chaotropic salt, DNA fragments selectively bind to the silica-based membrane in the spin column. Through a series of rapid washing and centrifugation steps, impurities such as primers, nucleotides, proteins, and enzymes are removed by the wash buffer. Finally, a low-salt, high-pH elution buffer is used to elute the purified DNA from the silica membrane.

V. Product Feature：

1. All silica membranes in the spin columns are made from high-quality, specially engineered adsorption material, ensuring minimal variation in binding capacity between columns.

2. The high-quality lysis buffer contains no sodium iodide or perchlorate commonly found in traditional formulations, thus avoiding inhibition of downstream enzymatic reactions such as digestion, ligation, and cloning after recovery.

3. The lysis buffer Buffer DD is tinted with phenol red, providing a yellow color that facilitates visual monitoring of lysis progress and pH changes, thereby optimizing binding conditions and significantly improving recovery efficiency.

4. The improved lysis buffer Buffer DD formulation greatly enhances buffering capacity and stability, maintaining the pH within the optimal binding range even with highly variable sample types.

5. The procedure is rapid and convenient, eliminating the need for toxic reagents such as phenol and chloroform, as well as ethanol precipitation.

VI. Application：

This product is suitable for recovering DNA fragments ranging from 100 bp to 5 kb from agarose gels containing 1–15 μg of DNA fragments. The recovery rate can reach up to 85%. The recovered DNA fragments are suitable for various routine experiments, such as enzymatic digestion, ligation, transformation, PCR template preparation, sequencing, and library construction.

VII. Precautions：

1. This product is intended for research use only. It is not authorized for application in medicine, clinical diagnostics, food, cosmetics, or related purposes.

2. The lysis buffer contains irritating compounds. Latex gloves should be worn during operation to avoid contact with skin, eyes, or clothing. In case of contact with skin or eyes, rinse immediately with plenty of water or physiological saline.

3. The typical recovery range of purified DNA fragments is between 100 bp and 40 kb. Recovery efficiency decreases significantly for fragments shorter or longer than this range.

4. The amount of DNA recovered depends on the initial DNA quantity, elution volume, and DNA fragment size. Typically, for DNA fragments ranging from 100 bp to 5 kb, with an input of 1–15 μg, the recovery rate can reach up to 85%.

5. When excising gel bands for DNA recovery, exposure to UV light can damage DNA fragments. It is recommended to use low-energy, long-wavelength UV light as much as possible and minimize the duration of exposure under UV light.

6. Elution Buffer EB does not contain the chelating agent EDTA and will not affect downstream enzymatic digestion, ligation, or other reactions. Water may also be used for elution, but ensure its pH is >7.5, as excessively low pH will reduce elution efficiency. DNA eluted with water should be stored at –20°C. For long-term storage, DNA may be eluted with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0); however, EDTA may interfere with downstream enzymatic digestion. If used, appropriate dilution is recommended.

7. Regarding the Use of Buffer BL 

1. During prolonged storage, the silica membrane in nucleic acid-binding spin columns may react with airborne charges or dust, which can reduce its nucleic acid binding capacity. Pretreating the column with Buffer BL significantly decreases the hydrophobic groups on the silica membrane and enhances its nucleic acid binding ability, thereby improving DNA recovery yield or efficiency. The conditioning solution is a strong alkaline solution. In case of accidental contact, rinse thoroughly with plenty of tap water. Always tighten the bottle cap after use to avoid exposure to air. Store at room temperature. Precipitation may form during storage; if present, heat at 37°C until the precipitate completely dissolves before use.
2. Procedure: Place a new silica membrane spin column into a collection tube. Pipette 100 μL of Buffer BL into the column. Centrifuge at 13,000 rpm for 1 minute. Discard the flow-through from the collection tube and return the spin column to the same tube. The column is now ready for use. Proceed with the subsequent steps.

VIII. Procedure (Please read the precautions before starting the experiment):

Note: Before initial use, add the specified amount of anhydrous ethanol to Wash Buffer WB and mix thoroughly. After adding, promptly check the box to mark that ethanol has been added, to avoid repeated addition.

1. Under long-wavelength UV light, use a clean blade to excise the target DNA band. Remove as much gel without DNA as possible to minimize the final gel volume.

2. Transfer the excised gel slice containing the DNA band into a 1.5 mL centrifuge tube and weigh it.

First weigh an empty 1.5 mL tube, then weigh again after adding the gel slice. Subtract the two weights to obtain the weight of the gel.

3. Add 3 volumes of Lysis Buffer DD to the gel.
For example, if the gel weighs 100 mg (which can be considered approximately 100 μL in volume), add 300 μL of Lysis Buffer.

If the gel concentration exceeds 2%, add 6 volumes of Lysis Buffer.

4. Incubate at 56°C for 10 minutes (or until the gel is completely dissolved). Vortex every 2–3 minutes to facilitate and accelerate dissolution.

5. Optional (usually not required): Add 150 μL of isopropanol per 100 mg of initial gel weight, and vortex to mix thoroughly.

In some cases, adding isopropanol may improve recovery efficiency. Do not centrifuge after adding isopropanol.

For recovering fragments larger than 4 kb, isopropanol should not be added, as it may reduce recovery efficiency.

Column Conditioning with Buffer BL:

Pretreating the silica membrane spin column with Buffer BL is a mandatory step. For the specific method, please refer to the previous section "Regarding the Use of Column Conditioning Solution".

6. Transfer the solution obtained from the previous step into the Spin EC column (placed in a collection tube). Let it stand at room temperature for 1 minute, then centrifuge at 12,000 rpm for 30–60 seconds. Discard the flow-through in the collection tube.

If the total volume exceeds 750 μL, the solution may be loaded onto the same Spin EC column in two separate batches.

The filtered lysis buffer may mix with residual strongly alkaline conditioning solution remaining in the collection tube, causing the color to change from yellow to orange-red or even purple. This is a normal color shift of the phenol red pH indicator under alkaline conditions.

7. Add 600 μL of Wash Buffer WB, then centrifuge at 12,000 rpm for 30 seconds and discard the flow-through.

8. Transfer the Spin EC column into a clean centrifuge tube. Apply 50 μL of Elution Buffer EB (pre-warmed in a 65–70°C water bath for better results) directly onto the center of the adsorption membrane. Let it stand at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 1 minute. To obtain a larger amount of DNA, the eluate may be reloaded onto the same column and centrifuged for another minute.

A larger elution volume generally results in higher elution efficiency. If a higher DNA concentration is required, the elution volume may be appropriately reduced, but the minimum volume should not be less than 25 μL. Excessively small volumes will reduce DNA elution efficiency and lower the final yield.