Plasmid Maxiprep Kit

Product Information

Product Description/Introduction

This kit adopts the classic alkaline lysis method and a technique where silica membranes reversibly bind to nucleic acids. It can extract 500 µg-1500 µg of plasmid DNA from 100 mL of overnight-cultured Escherichia coli. The extracted plasmid DNA can be directly used in various molecular biology experiments such as restriction enzyme digestion reaction, ligation reaction, PCR amplification, sequencing, and transformation.

Storage and Shipping Conditions

RNase A is shipped with wet ice and stored at -20°C. Other reagents are shipped and stored at room temperature, valid for 12 months.

Product Content

Before starting (please read carefully)

1. Bring your own anhydrous ethanol and 50 mL Nuclease-free centrifuge tubes.

2. Add all the RNase A provided in the kit to Buffer P1 before use, and it can be stored at 2-8°C for 6 months.

3. Please add 75 mL of anhydrous ethanol to Buffer PD and add 163 mL anhydrous ethanol to Buffer PW before first use.

4. If Buffer P2 precipitates, please heat it in a water bath at 37°C for a few minutes to restore clarification. After using Buffer P2, the lid should be closed immediately to avoid long-term contact with air.

5. Please pre-cool Buffer P3 at 4°C before use.

Assay Protocol/Procedures

1. Column balance: Add 2.5 mL of Buffer BL to the HiBind DNA Column, centrifuge at 10,000×g for 2 minutes, discard the waste liquid, and place the HiBind DNA Column back into the Collection Tube (the treated column is best used on the same day).

2. Take 100 mL of overnight-cultured bacterial solution, centrifuge at 10,000×g at room temperature for 1 minute to collect bacterial cells in a 50 mL centrifuge tube (remove as much of the supernatant as possible). For low-copy plasmids or plasmid DNA larger than 10 kb, it is recommended to collect bacterial cells from 200 mL of overnight culture and increase the volumes of Buffer P1, Buffer P2, and Buffer P3 by an equal volume.

3. Add 8 mL of Buffer P1 (please first check if RNase A has been added), and thoroughly resuspend the bacterial cells using a pipette or vortex mixer (the bacterial cells must be completely dispersed; otherwise, lysis will be affected, resulting in low quality and purity of the extracted plasmid).

4. Add 8 mL of Buffer P2 and immediately invert gently up and down 8-10 times to fully lyse the bacterial cells (do not shake violently in this step to avoid shearing and breaking of genomic DNA, and this step must be completed within 5 minutes). At this point, the solution should become clear and viscous. If it does not become clear, it may be due to an excessive amount of bacterial cells leading to incomplete lysis; in this case, invert gently another 5-8 times until the solution becomes completely clear.

5. Add 8 mL of pre-chilled (4°C) Buffer P3, immediately invert gently up and down 10-12 times to mix thoroughly. Tight clumps will form in the solution. Centrifuge at 10,000×g for 10-15 minutes. Slowly pour all the supernatant into the Column Filters, push the plunger to filter, and collect the filtrate in a nuclease-free 50 mL centrifuge tube (prepared by user).

6. Add anhydrous ethanol equal to 0.5 times the volume of the above solution, invert up and down to mix, then transfer the solution to the HiBind DNA Column (do not add more than 10 mL of liquid each time).

7. Centrifuge at 10,000×g at room temperature for 2 minutes, discard the waste liquid in the Collection Tube, and place the HiBind DNA Column back into the Collection Tube (the solution from Step 6 needs to be passed through the column multiple times).

8. Add 10 mL of Buffer PD (confirm whether anhydrous ethanol has been added) to the adsorption column, centrifuge at 10,000×g for 2 minutes, discard the waste liquid in the Collection Tube, and place the HiBind DNA Column back into the Collection Tube.

9. Add 10 mL of Buffer PW (confirm whether anhydrous ethanol has been added) to the HiBind DNA Column, centrifuge at 10,000×g for 2 minutes, discard the waste liquid in the Collection Tube, and place the HiBind DNA Column back into the Collection Tube.

10. Repeat Step 9.

11. Centrifuge at 10,000×g for 5 minutes, place the HiBind DNA Column in a new 50 mL Collection Tube, leave the lid open, and let it stand at room temperature for 10 minutes to allow complete evaporation of ethanol.

12. Add 1-1.5 mL of Buffer TE or nuclease-free water dropwise to the middle of the membrane in the HiBind DNA Column, let it stand at room temperature for 5 minutes, then centrifuge at 10,000×g for 5 minutes. To increase plasmid recovery efficiency, the obtained solution can be re-added to the HiBind DNA Columns, left to stand at room temperature for 5 minutes, and then centrifuged at 10,000×g for 5 minutes.

13. The resulting plasmid DNA can be stored long-term at -20°C.

Note

1. Please read the Product Manual carefully before use.

2. For low-copy plasmids or plasmid DNA larger than 10 kb, it is recommended to collect bacterial cells from 200 mL of overnight culture and increase the volumes of Buffer P1, Buffer P2, and Buffer P3 by an equal volume.

3. The eluate can be preheated to 60-65°C and the incubation time can be extended, which can improve the elution efficiency.

4. Before eluting the plasmid, ethanol should be completely evaporated to prevent residual ethanol from affecting subsequent downstream experiments.

5. For your safety and health, please wear a lab coat and disposable gloves during the operation.

For Research Use Only!

Flowchart