DNA Fragment Purification Kit Spin Column Method 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. It utilizes a high-quality lysis buffer that 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 unique formulation of Buffer DB integrates both lysis and binding functions, enabling a single kit to be used for various applications such as DNA recovery from agarose gels, cleanup and purification of PCR products, and purification and recovery of enzymatic digestion products. This eliminates the need to purchase multiple specialized kits, resulting in cost savings.

4. The buffer DB is tinted yellow to facilitate visual monitoring of lysis progress and pH changes, thereby achieving optimal binding conditions and significantly improving recovery efficiency.

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

6. 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 DNA recovery from agarose gels, purification and recovery of PCR reaction products, purification and recovery of enzymatically digested DNA fragments, purification after probe labeling, DNA sample concentration, and other related applications.

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. All centrifugation steps should be performed at room temperature using a conventional benchtop centrifuge capable of reaching 13,000 rpm.

3. The binding 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.

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

5. The amount of DNA recovered depends on the initial DNA quantity, elution volume, and DNA fragment size. Under general conditions, for DNA fragments ranging from 1–15 μg and 100 bp–5 kb, the recovery rate can be as high as 95%.

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. Introduction: 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) DNA recovery from agarose gels

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 Buffer DB.

If the gel weighs 100 mg, its volume can be considered as approximately 100 μL; accordingly, 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.

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

7. Add 600 μL of Wash Buffer WB (please check whether anhydrous ethanol has been added first!), centrifuge at 12,000 rpm for 30 seconds, and discard the flow-through

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

9. Place the Spin EC column back into the empty collection tube and centrifuge at 12,000 rpm for 2 minutes to thoroughly remove residual wash buffer, as any remaining ethanol in the wash buffer may inhibit downstream reactions.

10. Transfer the Spin EC column to 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. Allow it to stand at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 1 minute. If a higher DNA yield is desired, the eluate may be reloaded onto the same column and centrifuged for an additional 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; however, the minimum volume should not be less than 25 μL, as an excessively small volume will lower DNA elution efficiency and reduce the overall yield.

(2) DNA Purification for PCR Products or Digested Fragments：

1. For every 100 μL of post-PCR amplification mixture or post-digestion mixture, add 500 μL of Lysis/Binding Buffer DB and mix thoroughly. (If the initial volume is less than 100 μL, adjust it to 100 μL with double-distilled water beforehand.)

2. Transfer the solution from the previous step into a 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, and discard the flow-through in the collection tube.

3. From this step onward, the procedure is exactly the same as steps 7–10 for DNA recovery from agarose gels. Please refer to steps 7–10 of the "DNA Recovery from Agarose Gels" protocol.