8. Endotoxin-Free Plasmid DNA Maxi Extraction Kit, 500-1800 μg DNA; 10T $399,

Description

Product Description

This kit is designed for the extraction of endotoxin-free plasmid DNA from cultured bacteria. The kit utilizes a unique silica membrane adsorption technology in combination with endotoxin removal solutions Buffer ER and Buffer ED, enabling the isolation of high-purity plasmid DNA with concentrations ranging from 500 to 1800 μg while effectively removing endotoxins. The plasmid DNA extracted using this kit can be applied in various biological experiments, including restriction enzyme digestion, ligation reactions, PCR amplification, sequencing, transformation, transfection into different cells, and more.

Storage and Transportation

RNase A is transported with wet ice and should be stored at -20°C. Other reagents can be transported and stored at room temperature. The kit has a shelf life of 12 months.

Preparation before Use

1. Prepare an adequate amount of ethanol and Nuclease-free 50 mL centrifuge tubes.
2. Prior to use, add all of the RNase A to Buffer P1 and store at 4°C for up to 6 months.
3. Before using Buffer PW, add 163 mL of ethanol.
4. If Buffer P2 shows precipitation, heat it in a water bath at 37°C for a few minutes to restore clarity. After use, immediately close the lid to avoid prolonged exposure to air.
5. Pre-cool Buffer P3 at 4°C before use.

Experimental Procedure

1. Column equilibration step: Add 2.5 mL of Buffer BL to the HiBind DNA Column (previously placed in a 50 mL Collection Tube). Centrifuge at 8000 rpm (~8228×g) at room temperature for 2 minutes, discard the waste liquid, and return the HiBind DNA Column to the Collection Tube (it is best to use the processed column immediately).
2. Take 100 mL of overnight bacterial culture (for low-copy plasmids, it is recommended to use 200 mL of overnight bacterial culture). Centrifuge at 8000 rpm (~8228×g) at room temperature for 3 minutes to collect the bacterial cells in a 50 mL centrifuge tube (remove as much supernatant as possible).
3. Add 8 mL of Buffer P1 (check if RNase A has been added), thoroughly resuspend the bacterial cells using a pipette or vortex mixer (ensure complete dispersion of the cells, as incomplete dispersion can affect lysis and result in lower quality and purity of the extracted plasmid).
4. Add 8 mL of Buffer P2 and gently invert 8-10 times to fully lyse the cells (do not vigorously shake and ensure completion within 5 minutes). At this stage, the solution should become clear and viscous. If it does not become clear, it may indicate an excess of bacterial cells. Repeat gentle inversion until the solution becomes transparent.
5. Add 8 mL of Buffer P3 (pre-cooled at 4°C) and gently invert 10-12 times until the solution forms a compact aggregate. Place on ice for 5 minutes, centrifuge at 8000 rpm (~8228×g) at 4°C for 15 minutes, carefully pour all of the supernatant into the Column Filter, push the plunger to filter, and collect the filtrate in a Nuclease-free 50 mL centrifuge tube (provided by the user).
6. Add 2.4 mL of Buffer ER, gently mix by inverting, and leave on ice for 10 minutes (invert 3-5 times during this period). The solution should turn transparent blue.
7. Add 0.5 times the volume of the above solution of ethanol, invert gently, and transfer to the HiBind DNA Column (do not exceed 10 mL of liquid each time).
8. Centrifuge at 8000 rpm (~8228×g) at room temperature for 2 minutes, discard the waste liquid from the HiBind DNA Column, and return the HiBind DNA Column to the Collection Tube (the solution from step seven can be passed through the column multiple times).
9. Add 10 mL of Buffer ED to the HiBind DNA Column, centrifuge at 8000 rpm (~8228×g) at room temperature for 2 minutes, discard the waste liquid from the HiBind DNA Column, and return the HiBind DNA Column to the Collection Tube.
10. Add 10 mL of Buffer PW to the HiBind DNA Column, centrifuge at 8000 rpm (~8228×g) at room temperature for 2 minutes, discard the waste liquid from the collection tube, and return the HiBind DNA Column to the Collection Tube.
11. Repeat step 10.
12. Centrifuge at 8000 rpm (~8228×g) at room temperature for 5 minutes, then leave the HiBind DNA Column uncovered at room temperature for 10 minutes to allow ethanol to evaporate completely.
13. Place the HiBind DNA Column in a new 50 mL Collection Tube, suspend 1-2 mL of Buffer TE in the middle of the adsorption membrane, leave at room temperature for 5 minutes, and centrifuge at 8000 rpm (~8228×g) for 5 minutes. If higher plasmid recovery efficiency is desired, the obtained solution can be added back to the HiBind DNA Column, left at room temperature for 5 minutes, and centrifuged at 8000 rpm (~8228×g) for 5 minutes.
14. Transfer all elution solution from the 50 mL Collection Tube to a Nuclease-free 1.5 mL centrifuge tube and store at -20°C.

Precautions

1. Before proceeding, please read the product manual carefully.
2. When using the Column Filter, do not add excessive precipitates to avoid clogging the filter.
3. For plasmid DNA larger than 10 kb, increase the amount of bacterial cells collected.
4. Ensure complete evaporation of ethanol before eluting the plasmid to avoid residual ethanol affecting downstream experiments.
5. For your safety and health, wear lab coat and disposable gloves during the operation.

This product is for research use only and is not intended for clinical diagnosis!

(Product packaging may vary, please refer to the actual product.)