Product description

The Host Cell Residual DNA Sample Preparation Kit (Magnetic Bead Method) is designed for sample preparation prior to residual DNA detection in biological products and enables stable and efficient recovery of trace cellular DNA. The kit is compatible with various matrix buffers and can be used together with host cell residual DNA detection kits for different host cells, including CHO and E. coli.

Detection principle

This kit uses a magnetic bead method for rapid and efficient nucleic acid isolation. By changing the liquid-phase conditions, nucleic acids are specifically adsorbed onto the surface of magnetic beads coated with a special polymer. After the sample is lysed, the released nucleic acids bind to the magnetic beads. Impurities and inhibitors are then removed with wash buffers, and the purified nucleic acids are finally eluted from the magnetic beads with elution buffer.

Product components

Transportation and storage

Unopened reagents can be stored at room temperature (10°C to 30°C) for 12 months.

Once opened, the reagents should be used within 2 months. Keep all components tightly sealed at room temperature to prevent evaporation.

Required consumables and equipment

• Constant-temperature heating device

• Centrifuge

• Vortex mixer

• Magnetic rack

• Nuclease-free centrifuge tubes

• Nuclease-free pipette tips

Sample preparation

1. Intermediate samples from the purification process of biological products (potentially containing high levels of DNA)

1.1 Dilute before purification: Dilute the sample appropriately with 1×PBS (pH 7.4, without Mg²⁺ or Ca²⁺) before purification.

1.2 Purify before dilution: Purify the sample first and then dilute it with 1×PBS.

1.3 To ensure that the measured value falls within the linear range of the standard curve, samples with high DNA content should be diluted 100-fold or 1,000-fold. If the sample is diluted, use the diluent as the negative control.

2. Lyophilized powder samples (select one method)

2.1 Sample dissolution and purification: Dissolve the lyophilized sample with an appropriate diluent and then purify the sample.

2.2 Sample dissolution, dilution, and purification: Dissolve the lyophilized sample with an appropriate diluent to prepare a high-concentration solution, dilute the solution with the diluent, and then purify the sample.

2.3 It is recommended to dilute the lyophilized sample to 10 mg/mL or 100 mg/mL.

3. Samples with excessively high or low pH

3.1 In general, intermediate samples from biological product purification processes are neutral. Samples that are too acidic or alkaline may affect purification efficiency.

3.2 pH adjustment: Measure the sample pH before processing. Adjust the pH to neutral (pH 6.0-8.0) with 1 M HCl or NaOH before purification.

4. Control sample preparation

4.1 Parallel sample processing: To ensure the accuracy of the results, three independent DNA extractions and measurements are recommended for each sample.

4.2 Negative control (NTC): A negative control must be included in every experiment. Process the NTC together with the test samples to monitor cross-contamination or environmental contamination during sample preparation.

4.3 Extraction recovery control (ERC): Use an ERC to evaluate DNA extraction efficiency, recovery, and accuracy, and to assess the analytical method and system performance. The amount of spiked DNA should preferably be 2-10 times the unspiked measured value.

Experimental procedure

Manual extraction workflow

1. Remove all components from the kit, bring them to room temperature, and mix thoroughly before use. If a small amount of crystallization is observed in the Lysis Buffer, dissolve it completely before use.

2. Pretreatment: Prepare the working solution for each sample using 500 µL Lysis Buffer, 16 µL Magnetic Beads, and 20 µL Proteinase K.

3. Add 536 µL working solution and 100 µL sample to a 1.5 mL centrifuge tube. Vortex thoroughly, and then heat at 70°C for 5 minutes in a dry bath or metal bath.

4. After heating, centrifuge the tube for 5-10 seconds and place it on a magnetic rack for 2 minutes. Discard the supernatant. Avoid disturbing the magnetic beads on the tube wall when removing the supernatant.

5. Add 600 µL Wash Buffer I, close the tube, and vortex thoroughly. After a brief centrifugation, place the tube on a magnetic rack for 2 minutes and discard the supernatant.

6. Add 600 µL Wash Buffer II, close the tube, and vortex thoroughly. After a brief centrifugation, place the tube on a magnetic rack for 2 minutes and discard the supernatant.

7. To ensure complete removal of residual liquid, briefly centrifuge the tube again after Step 6 and place it on the magnetic rack for 2 minutes. Remove any residual liquid from the bottom of the tube, open the cap, and air-dry at room temperature for 1-3 minutes.

8. Add 100 µL Elution Buffer, close the tube, vortex for 5-10 seconds, and briefly centrifuge.

9. Heat the tube at 85°C for 4 minutes in a dry bath or metal bath.

10. Centrifuge the tube for 30 seconds and place it on the magnetic rack. Transfer the supernatant, which contains the purified nucleic acid, for subsequent use. For short-term storage, keep it at -25°C to -15°C. For long-term storage, keep it at -70°C or below.

Semi-automatic extraction workflow (manual loading into a deep-well plate)

1. Remove all components from the kit, bring them to room temperature, and mix thoroughly before use. If a small amount of crystallization is observed in the Lysis Buffer, dissolve it completely before use.

2. Preload the appropriate solutions according to the positions of the heating bars in the extraction instrument. Using a Pure 32 automatic nucleic acid extraction and purification instrument as an example, with heating bars located at columns 2, 6, 8, and 12, add the components as shown below.

Columns 1 and 7: add 16 µL Magnetic Beads.

Columns 2 and 8: add 500 µL Lysis Buffer.

Columns 3 and 9: add 600 µL Wash Buffer I.

Columns 4 and 10: add 600 µL Wash Buffer II.

Columns 6 and 12: add 100 µL Elution Buffer.

Note: If the heating bars of the extraction instrument are located at columns 1, 6, 7, and 8, exchange the positions of the Lysis Buffer and Magnetic Beads.

3. Add 20 µL Proteinase K and 100 µL test sample to the wells in columns 2 and 8 of the preloaded deep-well plate. Each plate can process 16 samples.

4. Place the deep-well plate into the instrument base, insert the 8-strip magnetic rod sleeves into their slots, select the program, and start the extraction.

Note: Even when extracting only one sample, install two 8-strip magnetic rod sleeves to prevent direct contact between the magnetic rods and the reagents.

Note: Place the deep-well plate with column 1 on the left and column 12 on the right. Incorrect orientation will prevent DNA recovery.

Extraction program using a Pure 32 instrument:

Note: Set the volume parameters according to the actual extraction performance; they may not exactly match the reagent volumes. Before starting the program, carefully check that the 8-strip magnetic rod sleeves are installed in the correct positions. If the heating bars of the extraction instrument are located at columns 1, 6, 7, and 8, set the well positions for Steps 1 and 6 to column 2 and set the well position for Step 2 (Lysis) to column 1.

8. The extracted products are located in columns 6 and 12. If they will not be used immediately, transfer them to sterile, nuclease-free 1.5 mL centrifuge tubes and store at -25°C to -15°C. For long-term storage, keep them at -70°C or below.

Precautions

1. Several buffers in this kit contain irritating guanidine salts. Always wear gloves and follow standard safety precautions. Avoid contact with skin, eyes, or mucous membranes. If contact occurs, rinse immediately with plenty of water and seek medical attention.

2. If a solution precipitates because of low temperature, incubate it in a 30°C water bath until the precipitate is completely dissolved before use.

Key points

1. During manual extraction, briefly centrifuge after each vortexing step in the DNA wash and elution procedures to ensure that no magnetic beads or liquid remain on the tube cap or wall before magnetic separation.

2. During each liquid addition and removal step, gently open the tube cap with your thumb and avoid touching the inner ring beneath the cap. Prevent splashing when pipetting.

3. During magnetic separation, rotate the centrifuge tube as needed to concentrate the magnetic beads.

4. When drying after ethanol removal, observe the magnetic beads and avoid excessive drying, which may prevent complete resuspension during elution.

5. Whenever possible, perform the subsequent DNA detection on the same day as sample purification to ensure accurate results.

Troubleshooting