Plasmid Midiprep Kit servicebio G3649-10T
$50.00
In stock
SKU
G3649-10T
Product Information
| Product Name: Plasmid Midiprep Kit | Cat. No.: G3649-10T |
| Spec.: 10T |
Product Description/Introduction
This kit adopts the classic alkaline lysis method and a purification approach where glass fiber membranes bind to plasmids efficiently and specifically. It is suitable for purifying high-purity plasmids from bacteria cultured in 20-50 mL of medium. Depending on the copy number of the plasmids, the yield ranges from 100 to 400 μg. The extracted plasmids can be directly used in various subsequent 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
| Component Number: G3649-1 | Component: Buffer BL |
| G3649-10T: 25mL | Component Number: G3649-2 |
| Component: Buffer P1 | G3649-10T: 30 mL (Add RNase A before use) |
| Component Number: G3649-3 | Component: Buffer P2 |
| G3649-10T: 30 mL | Component Number: G3649-4 |
| Component: Buffer P3 | G3649-10T: 30 mL |
| Component Number: G3649-5 | Component: RNase A |
| G3649-10T: 600 μL | Component Number: G3649-6 |
| Component: Buffer PD | G3649-10T: 16 mL (Add 24 mL of anhydrous ethanol before use) |
| Component Number: G3649-7 | Component: Buffer PW |
| G3649-10T: 16 mL (Add 64 mL of anhydrous ethanol before use) | Component Number: G3649-8 |
| Component: Buffer TE | G3649-10T: 15 mL |
| Component Number: G3649-9 | Component: Column Filters |
| G3649-10T: 10 pcs | Component Number: G3649-10 |
| Component: HiBind DNA Midi Columns | G3649-10T: 10 pcs |
| Component Number: G3649-11 | Component: 15 mL Centrifuge Tubes |
| G3649-10T: 10 pcs | Manual |
| 1 copy |
Before starting (please read carefully)
1. Prepare anhydrous ethanol by yourself.
2. Before use, add all RNase A into Buffer P1. The mixture can be stored at 2-8°C for 6 months.
3. Add 24 mL of anhydrous ethanol to Buffer PD before use.
4. Add 64 mL of anhydrous ethanol to Buffer PW before use.
5. If precipitation occurs in Buffer P2, heat it in a 37°C water bath for a few minutes to restore clarity. After use, tightly close the lid immediately to prevent the reagent from being in prolonged 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 (the treated column is best used immediately).
2. Take 20-50 mL of overnight cultured bacterial solution, centrifuge at 8000×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 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 check whether RNase A has been added), and thoroughly resuspend the bacterial cells using a pipette or vortex oscillator (the bacterial cells must be completely dispersed, otherwise, lysis will be affected, leading to low quality and purity of the extracted plasmid).
4. Add 2.5 mL of Buffer P2, 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 resulting in incomplete lysis, and the amount of bacterial cells should be reduced.
5. Add 2.5 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 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 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 Midi Column (do not add more than 4 mL of liquid each time).
7. 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 6 needs to be passed through the column multiple times).
8. Add 3.5 mL of Buffer PD (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.
9. Add 3.5 mL of Buffer PW (confirm whether absolute 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.
10. Repeat Step 9.
11. Centrifuge at 4500×g at room temperature for 10 minutes, then place the HiBind DNA Midi Column in a 15 mL Centrifuge Tube, leave the lid open, and let it stand at room temperature for 10 minutes to allow complete evaporation of ethanol.
12. Add 500-1000 μL of Buffer TE or nuclease-free water dropwise to the middle of the membrane in the HiBind DNA Midi Column, let it stand at room temperature for 5 minutes, then centrifuge at 4500×g for 5 minutes. To increase plasmid recovery efficiency, the obtained solution can be re-added to the HiBind DNA Midi Column, left to stand at room temperature for 5 minutes, and then centrifuged at 4500×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. When the extracted plasmid is a low-copy plasmid or has a size larger than 10 kb, the amount of bacterial cells collected should be increased, and the volumes of Buffer P1, Buffer P2, and Buffer P3 should be increased proportionally at the same time.
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 operation.
For Research Use Only!
Flowchart
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