Endo-Free Plasmid Midiprep Kit servicebio G3648-10T
$50.00
In stock
SKU
G3648-10T
Product Information
| Product Name: Endo-Free Plasmid Midiprep Kit | Cat. No.: G3648-10T |
| Spec.: 10T |
Product Description/Introduction
This kit utilizes a glass fiber membrane with specific adsorption capacity for plasmids, combined with a set of special buffer solutions. It enables efficient extraction of various endotoxin-free plasmids (endotoxin content≤0.1 EU/μg) from cultured bacteria. Depending on the copy number of the plasmid, the plasmid yield ranges from 100 to 400 μg. The high-purity extracted plasmids can be applied to various subsequent biological experiments, such as restriction enzyme digestion, ligation reaction, PCR amplification, sequencing, transformation, and transfection.
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
| Component Number: G3648-1 | Component: Buffer BL |
| G3648-10T: 25mL | Component Number: G3648-2 |
| Component: Buffer P1 | G3648-10T: 30 mL (Add RNase A before use) |
| Component Number: G3648-3 | Component: Buffer P2 |
| G3648-10T: 30 mL | Component Number: G3648-4 |
| Component: Buffer P3 | G3648-10T: 30 mL |
| Component Number: G3648-5 | Component: RNase A |
| G3648-10T: 600 μL | Component Number: G3648-6 |
| Component: Buffer ER | G3648-10T: 10 mL |
| Component Number: G3648-7 | Component: Buffer ED |
| G3648-10T: 40 mL | Component Number: G3648-8 |
| Component: Buffer PW | G3648-10T: 16 mL (Add 64 mL of anhydrous ethanol before use) |
| Component Number: G3648-9 | Component: Buffer TE |
| G3648-10T: 15 mL | Component Number: G3648-10 |
| Component: Column Filters | G3648-10T: 10 pcs |
| Component Number: G3648-11 | Component: HiBind DNA Midi Columns |
| G3648-10T: 10 pcs | Component Number: G3648-12 |
| Component: 15 mL Centrifuge Tubes | G3648-10T: 10 pcs |
| Manual | 1 copy |
Before starting (please read carefully)
1. Prepare anhydrous ethanol by yourself.
2. Prepare a 42°C water bath or heating block.
3. Before use, add all RNase A to Buffer P1. It can be stored at 2-8°C for 6 months.
4. Add 64 mL of anhydrous ethanol to Buffer PW before use.
5. 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.
6. Please pre-cool Buffer P3 at 4°C before use.
Assay Protocol/Procedures
1. Column balance: Add 2 mL of Buffer BL to the HiBind DNA Midi Column, centrifuge at 4500×g at room temperature for 2 minutes, discard the waste liquid, and place the HiBind DNA Midi Column back into the Collection Tube (it is recommended to use the treated column immediately).
2. Take 20-50 mL of overnight cultured bacterial solution, centrifuge at 8000×g at room temperature for 1 minute to collect the bacterial cells in a 50 mL centrifuge tube (remove the supernatant as completely as possible). For low-copy plasmids or plasmids larger than 10 kb, it is recommended to appropriately increase the amount of bacterial cells and proportionally increase the volumes of Buffer P1, Buffer P2, and Buffer P3.
3. Add 2.5 mL of Buffer P1 (please first confirm whether RNase A has been added), and thoroughly resuspend the bacterial cells using a pipette or vortex oscillator (it is essential to completely disperse the bacterial cells; otherwise, lysis will be affected, resulting in low quality and purity of the extracted plasmid).
4. Add 2.5 mL of Buffer P2, and immediately invert gently 8-10 times to fully lyse the bacterial cells (vigorous shaking is not allowed 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, and the amount of bacterial cells should be considered for reduction.
5. Add 2.5 mL of pre-chilled (at 4°C) Buffer P3, immediately invert gently 10-12 times to mix thoroughly, and a tight aggregate will form in the solution. Centrifuge at 8000×g at room temperature for 15 minutes. Slowly pour all the supernatant into the Column Filter, push the plunger to filter, and collect the filtrate in a 15 mL nuclease-free centrifuge tube (prepared by user).
6. Add 750 μL of Buffer ER, invert to mix well, then place on ice for 10 minutes (invert to mix 3-5 times during this period); the solution will turn transparent blue.
7. Incubate at 42°C for 5 minutes until the solution becomes turbid again. Centrifuge at 4500×g at room temperature for 5 minutes (the centrifuge temperature must return to room temperature; otherwise, the stratification will be affected). At this point, the solution will separate into two layers (if the stratification is not obvious, leave it at room temperature for 1-3 minutes). The upper layer is a clear aqueous phase, and the lower layer is blue. Carefully collect the upper aqueous phase into a new 15 mL nuclease-free centrifuge tube (prepared by the user).
8. Add anhydrous ethanol in an amount equal to 0.5 times the volume of the aforementioned solution, invert up and down to mix well, then transfer the solution to the HiBind DNA Midi Column (do not add more than 4 mL of liquid each time).
9. Centrifuge at 4500×g at room temperature for 3 minutes, discard the waste liquid in the Collection Tube, and place the HiBind DNA Midi Column back into the Collection Tube (the solution from Step 8 needs to pass through the column multiple times).
10. Add 3.5 mL of Buffer ED to the adsorption column, centrifuge at 4500×g at room temperature for 3 minutes, discard the waste liquid in the Collection Tube, and place the HiBind DNA Midi Column back into the Collection Tube.
11. Add 3.5 mL of Buffer PW (confirm whether anhydrous ethanol has been added) to the HiBind DNA Midi Column, centrifuge at 4500×g at room temperature for 3 minutes, discard the waste liquid in the Collection Tube, and place the HiBind DNA Midi Column back into the Collection Tube.
12. Repeat Step 11.
13. Centrifuge at 4500×g at room temperature for 10 minutes. Then place the HiBind DNA Midi Column into a 15 mL Centrifuge Tube, leave the lid open, and let it stand at room temperature for 10 minutes to allow the ethanol to evaporate completely.
14. Add 500-1000 μL of Buffer TE or Endo-free Water to the middle area of the membrane in the HiBind DNA Midi Column, and let it stand at room temperature for 5 minutes, followed by centrifugation at 4500×g for 5 minutes. If you need to improve the plasmid recovery efficiency, the collected solution can be re-added to the HiBind DNA Midi Column. After standing at room temperature for 5 minutes, centrifuge again at 4500×g for 5 minutes.
15. The obtained plasmid DNA can be stored at -20°C for long-term preservation.
Note
1. Please read the Product Manual carefully before use.
2. Avoid direct contact with Buffer P2 and Buffer P3. Tightly close the lid immediately after using Buffer ED.
3. When the extracted plasmid is a low-copy plasmid or larger than 10 kb, increase the amount of bacterial cells collected and proportionally increase the volumes of Buffer P1, Buffer P2, and Buffer P3.
4. The elution efficiency can be improved by preheating the eluent to 60-65°C and extending the elution incubation time.
5. Before eluting the plasmid, ensure that ethanol is completely evaporated to prevent residual ethanol from affecting downstream experiments.
6. For your safety and health, please wear a lab coat and disposable gloves during operation.
For Research Use Only!
Flowchart
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