Endo-Free Plasmid DNA Miniprep Kit servicebio G3646-50T
$50.00
In stock
SKU
G3646-50T
Product Information
| Product Name: Endo-Free Plasmid DNA Miniprep Kit | Cat. No.: G3646-50T |
| Spec.: 50T |
Product Description/Introduction
The kit uses a specially formulated buffer solution, combined with the reversible adsorption technology of nucleic acid by silica gel membrane, which can almost completely remove endotoxin (≤0.1 EU/μg), protein and other impurities from 1-5 mL overnight cultured Escherichia coli. The yield of plasmid DNA is 5-20 μg. The extracted plasmid can be used in biological experiments such as enzyme digestion reactions, ligation reactions, PCR amplification, sequencing, transformation, transfection, etc.
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: G3646-1 | Component: Buffer BL |
| G3646-50T: 30 mL | Component Number: G3646-2 |
| Component: Buffer P1 | G3646-50T: 15 mL (Add RNase A before use) |
| Component Number: G3646-3 | Component: Buffer P2 |
| G3646-50T: 15 mL | Component Number: G3646-4 |
| Component: Buffer P3 | G3646-50T: 15 mL |
| Component Number: G3646-5 | Component: RNase A |
| G3646-50T: 150 μL | Component Number: G3646-6 |
| Component: Buffer ER | G3646-50T: 4 mL |
| Component Number: G3646-7 | Component: Buffer ED |
| G3646-50T: 35 mL | Component Number: G3646-8 |
| Component: Buffer PW | G3646-50T: 15 mL (Add 60 mL of anhydrous ethanol before use) |
| Component Number: G3646-9 | Component: Buffer TE |
| G3646-50T: 10 mL | Component Number: G3646-10 |
| Component: HiBind DNA Mini Columns (with Collection Tubes) | G3646-50T: 50 pcs |
| Manual | 1 copy |
Before starting (please read carefully)
1. Bring your own anhydrous ethanol and 1.5 mL Nuclease-free centrifuge tubes.
2. Prepare 42℃ water bath or heating module.
3. Before use, add all the RNase A to Buffer P1 and it can be stored at 4°C for 6 months.
4. Please add 60 mL 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 500 μL Buffer BL to HiBind DNA Mini Column, centrifuge at 12,000 rpm for 1 minute at room temperature, discard the filtrate, and put the HiBind DNA Column back into the Collection Tube (The treated column is best used immediately).
2. Take 1-5 mL of overnight culture bacterial solution, centrifuge at 12,000 rpm for 1 minute at room temperature to collect the bacteria in a 1.5 mL centrifuge tube (try to remove all the supernatant).
3. Add 250 μL of Buffer P1 (Please check if RNase A is added first), and use a pipette or vortex oscillator to thoroughly suspend the bacteria (Be sure to disperse the bacteria thoroughly, otherwise it will affect the lysis, resulting in low quality and purity of the extracted plasmid).
4. Add 250 μL of Buffer P2, immediately and gently turn it upside down for 8-10 times to make the bacteria fully lysed (This step should not be violently shaken to avoid genomic DNA shearing and breakage, and should be done within 5 minutes). At this point, the solution should become clear and sticky, if it does not become clear, it may be that there are too many bacteria and the lysis is not complete, and consideration should be given to reducing the amount of bacteria.
5. Add 250 μL of Buffer P3 (pre-cool in advance), immediately and gently turn it upside down 10-12 times, the solution appears compact agglomerate, centrifuge at 12,000 rpm for 10 minutes, transfer the supernatant to a new 1.5 mL centrifuge tube.
6. Add 75 μL of Buffer ER, mix by inverting 3-5 times, then incubate on ice for 10 minutes (mix by inverting 3-5 times during this period), the solution will appear transparent blue.
7. Incubate at 42°C for 5 minutes, the solution recovers turbidity. Centrifuge at 12,000 rpm for 10 minutes at room temperature (The centrifuge temperature must be restored to room temperature, otherwise it will affect stratification), the solution is divided into upper and lower layers (If the stratification is not obvious, leave at room temperature for 1-3 minutes), the upper layer is clear water phase and the lower layer is blue. Carefully collect the upper water phase into the new Nuclease-Free 1.5 mL centrifuge tubes.
8. Add anhydrous ethanol in an amount equal to 0.5 times the volume of the above solution, mix upside down and transfer to HiBind DNA Mini Column (no more than 700 μL each time), centrifuge at 12,000 rpm for 1 minute at room temperature, discard the filtrate in the Collection Tube. Put the HiBind DNA Mini Column back into the Collection Tube (the solution can pass through the column many times).
9. Add 600 μL of Buffer ED to the HiBind DNA Mini Column, centrifuge at 12,000 rpm for 1 minute at room temperature, discard the filtrate in the Collection Tube. Put the HiBind DNA Mini Column back into the Collection Tube.
10. Add 600 μL of Buffer PW to HiBind DNA Mini Column, centrifuge at 12,000 rpm for 1 minute at room temperature, discard the filtrate in the Collection Tube. Put the HiBind DNA Mini Column back into the Collection Tube.
11. Repeat step 10.
12. Centrifuge at 12,000 rpm for 2 minutes at room temperature, place the HiBind DNA Mini Column in a new Nuclease-Free 1.5 mL centrifuge Tube. Open the lid and leave it at room temperature for 5 min to thoroughly volatilize residual ethanol.
13. Add 50 μL of Buffer TE or Endo-Free Water to the membrane center of the HiBind DNA Mini Column and incubate at room temperature for 2 minutes. Centrifuge at 12,000 rpm for 2 minutes. If you need to increase the efficiency of plasmid recovery, the obtained solution can be re-added to HiBind DNA Mini Column, place at room temperature for 2 minutes and centrifuge at 12,000 rpm for 2 minutes.
14. The obtained plasmid DNA can be preserved for a long time at -20°C.
Note
1. Please read the Product Manual carefully before use.
2. When the copy number of the extracted plasmid is low or the plasmid fragment is larger than 10kb, the amount of bacteria collection should be increased, and the dosage of Buffer P1, Buffer P2 and Buffer P3 should be increased in equal proportion.
3. Ethanol should be completely volatilized before eluting the plasmid to avoid the influence of residual ethanol on downstream experiments.
4. Buffer TE can be preheated at 60-65°C and the elution incubation time can be prolonged to improve the elution efficiency.
5. For your safety and health, please wear a lab coat and disposable gloves.
For Research Use Only!
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