Endotoxin-free plasmid purification kit
32-minute rapid extraction: a revolution in detonation efficiency
One-click copy of information
Instruction manual- Product Description
- Composition
- Precautions
-
Product Overview
This product employs the classic alkaline lysis method, combined with column‑based silica‑membrane technology and an improved endotoxin‑removal solution, to isolate high‑quality plasmid DNA from 100–200 mL of overnight bacterial cultures. It effectively eliminates residual endotoxins, yielding highly pure plasmid DNA. The procedure is simple and rapid, enabling the quick extraction of 0.2–1.5 mg of plasmid DNA.
The residual endotoxin level in the extracted plasmid DNA is extremely low (＜0.1 EU/μg), making it suitable for direct use in cell transfection, animal injection, restriction digestion, PCR, sequencing, and other experiments.
Storage conditions
The solution L of this product should be stored at 2–8°C, while the other components should be stored at room temperature.
If RNase A has been completely added to Solution P1 and thoroughly mixed, Solution P1 should be stored at 2–8°C for a shelf life of 6 months.
Pre-experiment preparation
1. Before first use, add all of the RNase A to solution P1, mix thoroughly, and mark the tube.
2. Prepare a 2/50 mL centrifuge tube, 80% ethanol, and other necessary materials.
Instructions for Use
1. Pipette 100–200 mL of overnight‑grown bacterial culture into a 50 mL centrifuge tube, and centrifuge at 12,000 × g for 1 minute to pellet the cells. (If the culture volume is large, perform multiple centrifugations to collect all the cells in a single tube.) Carefully aspirate and discard the supernatant, ensuring that all liquid is removed.
Note: When culturing cells in enriched media such as 2×YT or TB, the volume of the culture should be appropriately reduced. If centrifuging at 8,000 × g, spin for 2 minutes; if centrifuging at 4,000 × g, spin for 3–5 minutes.
2. Sequentially add 8 mL of Solution P1 (which already contains RNase A) and 160 μL of Solution L, and vortex the cells until they are completely resuspended.
Note: If the bacterial cells are not completely resuspended and dispersed, it will affect the subsequent alkaline lysis step, thereby reducing the yield of plasmid DNA. Solution L can enhance plasmid extraction efficiency; please add 160 μL of Solution L to Solution P1 in the centrifuge tube first to avoid loss.
3. Add 8 mL of Solution P2, gently invert the tube 8 times, and let it stand at room temperature for 4 minutes (do not exceed 5 minutes).
Note: Do not vigorously vortex during this step; mix gently to avoid residual genomic DNA. Incubation time should not exceed 5 minutes to prevent plasmid degradation. At this point, the bacterial suspension should become clear and viscous. If it remains cloudy, it may indicate an excessive cell density or incomplete lysis; reduce the amount of cells used.
4. Add 8 mL of Solution P3, immediately invert the tube 15 times; a white flocculent precipitate should form. Allow to stand at room temperature for 5–10 minutes, then centrifuge at 12,000 × g for 8 minutes.
Note: After adding solution P3, mix immediately to prevent localized precipitation. If the pellet is not tightly compacted and contains a significant amount of supernatant, it indicates an excessive cell biomass; reduce the cell concentration accordingly. Use 8,000 × g for 10 minutes, or 4,000 × g for 12 minutes.
5. Transfer the supernatant to a new 50 mL centrifuge tube, add an endotoxin‑removal solution equal to 0.5× the volume of the supernatant, and invert the tube 15 times to ensure thorough mixing.
6. Transfer the mixture from Step 5 into the adsorption column (which has already been placed in the collection tube), centrifuge at 12,000 × g for 1 minute, and discard the supernatant.
Note: The maximum loading volume of the spin column is 14.5 mL. If the sample volume exceeds this limit, load it in multiple aliquots. If the centrifuge rotor is tilted at a significant angle, we recommend keeping the loaded volume below 12 mL. When using 4,000×g to 8,000×g, centrifuge for 1 minute.
7. Repeat step 6 until the entire mixture has been loaded onto the column.
8. Add 10 mL of protein‑removal solution to the adsorption column, let stand at room temperature for 1 min, centrifuge at 12,000 × g for 1 min, and discard the supernatant.
Note: If using 4,000 × g to 8,000 × g, centrifuge for 1 minute.
9. Add 10 mL of 80% ethanol to the adsorption column, let it stand at room temperature for 1 min, centrifuge at 12,000 × g for 1 min, and discard the supernatant.
Note: If using 4,000 × g to 8,000 × g, centrifuge for 1 minute.
10. Place the adsorption column back into the collection tube and centrifuge at 12,000 × g for 2 minutes.
Note: If using 8,000 × g, centrifuge for 3 minutes. If using 4,000 × g, centrifuge for 5 minutes.
11. Place the adsorption column into a new collection tube, add 1–2 mL of ddH₂O to the center of the membrane, let it stand at room temperature for 5 minutes, centrifuge at 12,000 × g for 3 minutes, and discard the adsorption column.
Note: If using 8,000 × g, centrifuge for 3 minutes. If using 4,000 × g, centrifuge for 15 minutes.
12. The extracted plasmid DNA was stored at −20°C.
-
Product Composition
Component Net weight/10 T Item number Solution P1 82 mL 400-204A Solution P2 82 mL 400-204B Solution P3 82 mL 400-204C Except for endotoxin solution 120 mL 400-204D Protein removal solution 105 mL 400-204E RNase A 1.64 mL 400-204F Solution L 1.64 mL 400-204G ddH2O 25 mL 400-204H Adsorption column 10 pieces 400-204I Collection tube 20 pieces 400-204J -
Precautions
1. Solution P2 may precipitate at low temperatures. If precipitation occurs, dissolve the precipitate by heating to 37°C until the solution becomes clear before use.
2. After use, each component should be promptly capped to prevent prolonged exposure to air, which may lead to alcohol volatilization and changes in pH.
Keywords
GOONIE
Previous
Next
Product Recommendations
Content is being updated.
Submit your message
If you are interested in our products, please provide your email address, and we will contact you as soon as possible. Thank you!
Goonie Biotechnology (Guangzhou) Co., Ltd.
Room 321–332, Building G5, No. 39 Ruihe Road, Huangpu District, Guangzhou
Customer Service QR Code
Friendly Links
