2. Universal (PCR products) DNA Purification and Gel DNA Purification Extraction Kit, 100T $ 128

Product Information

Description/Introduction

This kit is used for purifying DNA from TAE or TBE agarose gels, or recovering DNA form PCR products based on a special Bind DNA Mini Columns. The Buffer GL contains pH indicator, which can judge whether the gel solution or PCR product recovery has reached the optimal state according to the solution color. DNA fragments of 100 bp-10 kb can be recovered with a recovery rate of 85%. The amount of DNA adsorbed on each column was 20 μg. The DNA recovered using this kit can be used for a variety of routine procedures, including restriction enzyme digestion, PCR reaction, DNA sequencing, DNA library screening, and cloning.

Storage and Handling Conditions

Store and transport at room temperature, valid for 12 months.

Component

Assay Protocol / Procedures

Before Starting

1. Preparing Buffer PW with ethanol : add 60 mL 100% ethanol to the Buffer PW, then mark the bottle on the Buffer PW label to indicate that 100% ethanol was added. Store the Buffer PW with ethanol at room temperature, and tighten the cap after use to prevent volatilization.

2. (Optional) Column balance: Put the Bind DNA Mini Columns into the Collection Tubes, add 500 μL solution Buffer BL to the Bind DNA Mini Columns, centrifuge at 10,000 g for 1 min, remove the waste liquid in the Collection Tubes. Return the Bind DNA Mini Columns to the collection tube (please use the column treated on the day).

3. Maintain a sterile working environment when handling DNA to avoid any contamination from DNases.

4. Make sure all equipment that comes in contact with DNA is sterile, including pipette tips and tubes.

lGel extraction protocol

1. Excising the gel slice: After completing agerose gel electrophoresis, excise the area of the gel containing the desired DNA fragment using a clean, sharp razor blade. Minimize the amount of agarose surrounding the DNA fragment. Weigh the gel slice containing the DNA fragment using a scale sensitive to 0.001 g. place the gel slice into a 1.5-mL microcentrifuge tube (for≤2% agarose gels) or a 5.0-mL microcentrifuge tube(for≥2% agarose gels).

Note: The maximum amount of starting gel is≤400 mg per tube. If your gel slice exceeds 400 mg, cut the gel into smaller slices to ensure no one piece exceeds 400 mg. Place additional gel slices into separate microcentrfuge tubes. For this case, an additional Bind DNA Mini Column is required for each extra gel slice during the purification.

2. Dissolving the gel slice: Place≤400 mg of the excised gel containing DNA into microcentrifuge tube, add equal volume of Buffer GL to every 1 volume of gel (e.g., add 100 µL of Buffer GL for every 100 mg of gel). Place the tube containing the gel slice and Buffer GL into a 60℃ water bath or heat block and incubate at 60℃ for at least 10 min. Invert the tube every 3 min to ensure the complete gel dissolution.

Note: (1) High concentration gels (＞2% agarose) or large gel slices may take longer than 10 min to dissolve. (2) For optimal DNA yields, add half of gel volume isopropanol to the dissolved gel slice. (3) The best color of gel solution is faint yellow. If the color of the gel solution is red or violet, add about 10 µL of 3 M sodium acetate solution (pH 5.2) to adjust the gel solution to faint yellow.

3. Pipet the dissolved gel piece solution containing the DNA fragment into the center of a Bind DNA Mini Column inside a Collection Tube. Centrifuge the tube at 10,000×g for 1 min. Discard the flow-through and replace the Bind DNA Mini Column into the Collection Tube.

Note: Don not load >800 μL gel solution one time. If the total volume exceeds 800 µL, the gel solution can be devided into multiple to load.

4. Add 700 µL of Buffer PW (Please check whether 100% ethanol has been added before use) to the Bind DNA Mini Columns. Centrifuge the tube at 10,000×g for 1 min. Discard the flow-through and replace the column into the Collection tube.

Note: If the recovered DNA is used for salt-sensitive experiments, such as flat end joining experiment or direct sequencing, it is recommended to let the tube stand for 2-5 min after adding Buffer PW.

5. Again, add 700 µL of Buffer PW to the Bind DNA Mini Columns. Centrifuge the tube at 10,000×g for 1 min. Discard the flow-through and replace the column into the Collection tube.

6. Centrifuge the tube again at 10,000×g for 2-3 min to remove any residual Buffer PW and ethanol. Discard the Collection Tube and place the Bind DNA Mini Column into a new 1.5-mL centrifuge tube, let stand at room temperature for 5 min.

Note: Residual ethanol would affect subsequent enzyme digestion and PCR experiments.

7. Add 30-50 μL Elution Buffer or ddH₂O to the center of the Bind DNA Mini Column, incubate the tube at room temperature for 2 min. Centrifuge the tube at 10,000×g for 2 min. Then the 1.5-mL centrifuge tube contains the purified DNA. Discard the Bind DNA Mini Column.

Note: (1) Preheat the Elution Buffer or ddH₂O at 60-65℃ before use could increase the DNA elution effect. (2) The volume of Elution Buffer or ddH₂O should not be less than 30 μL to ensure the elution efficiency. (3) If the purified DNA is used for DNA sequencing, it is recommended to use ddH₂O for elution. (4)always use sterile water with pH 7.0-8.5, if you are using water for elution.

8. Store the purified DNA at 4℃ for immediate use or aliquot the DNA and store at -20℃ for long-term storage. Avoid repeated freezing and thawing of the DNA.

lPCR product purification protocol

1. Add 3 volume of Buffer GL to 1 volume of PCR reaction, mix well.

Note: (1) Maintain PCR volume of 50-100 μL. (2) For optimal DNA yields, add 3/4 volume of isopropanol to 1 volume of PCR reaction.

2. Add sample to the Bind DNA Mini Column (placed into the Collection Tube). centrfuge the tube at room temperature at 10,000×g for 1 min.

3. Discard the flow-through and replace the Bind DNA Mini Column into the Collection Tube.

4. Add 700 µL of Buffer PW (Please check whether anhydrous ethanol has been added before using) to the Bind DNA Mini Column. Centrifuge at 10,000×g for 1 min.

5. Discard the flow-through and place the Bind DNA Mini Column back into the Collection Tube.

Note: If the recovered DNA is used for salt-sensitive experiments, such as flat end joining experiment or direct sequencing, it is recommended to let the tube stand for 2-5 min after adding Buffer PW.

6. Again, add 700 µL of Buffer PW (Please check whether anhydrous ethanol has been added before using) to the Bind DNA Mini Column. Centrifuge at 10,000×g for 1 min.

7. Centrifuge the tube again at 10,000×g for 2-3 min to remove any residual Buffer PW and ethanol. Discard the Collection Tube and place the Bind DNA Mini Column into a new 1.5-mL centrifuge tube, let stand at room temperature for 5 min.

Note: Residual ethanol would affect subsequent enzyme digestion and PCR experiments.

8. Add 30-50 μL Elution Buffer or ddH₂O to the center of the Bind DNA Mini Column, incubate the tube at room temperature for 2 min. Centrifuge the tube at 10,000×g for 2 min. Then the 1.5-mL centrifuge tube contains the purified DNA. Discard the Bind DNA Mini Column.

Note: (1) Preheat the Elution Buffer or ddH₂O at 60-65℃ before use could increase the DNA elution effect. (2) The volume of Elution Buffer or ddH₂O should not be less than 30 μL to ensure the elution efficiency. (3) If the purified DNA is used for DNA sequencing, it is recommended to use ddH₂O for elution. (4)always use sterile water with pH 7.0-8.5, if you are using water for elution.

9. Store the purified DNA at 4℃ for immediate use or aliquot the DNA and store at -20℃ for long-term storage. Avoid repeated freezing and thawing of the DNA.

Notes 

1. In the column balancing step, the addition of Buffer BL can improve the adsorption capacity, homogeneity and stability of the Bind DNA Mini Columns, and eliminate the influence of high temperature, humidity or other adverse environmental factors on the Bind DNA Mini Columns. The column balancing step may also be omitted.

2. Please check whether the specified amount of anhydrous ethanol is added to Buffer PW before using.

3. Please tighten the lid in time to prevent solvent volatilization after each solution is used.

4. For your protection, always wear a laboratory coat, gloves, and safety glasses during the procedures.

For Research Use Only!

Ver. No.: V1.0-202102