Product Introduction:
This kit uses a novel silica-based membrane technology, coupled with an optimized buffer system, to allow plasmid DNA to bind efficiently and specifically to the membrane. It also utilizes a special buffer system and endotoxin removal filter column to effectively remove impurities such as endotoxins and proteins.
Each adsorption column can adsorb up to 40 μg of plasmid DNA. This kit is suitable for extracting 1-5 mL of bacterial solution. The extracted plasmid has high purity and stable quality. It is particularly suitable for cell transfection. It can also be used for DNA sequencing, PCR, and PCR-based
mutation, in vitro transcription, bacterial transformation, endonuclease digestion and other downstream experiments.
Product ingredients:
| Component name |
FS-B1703-S
|
FS-B1703-01
|
Storage temperature |
|
10 preps
|
50 preps
|
|
Resuspension Buffer P1
|
3 mL
|
15 mL
|
RT |
|
Lysis Buffer P2
|
3 mL
|
15 mL
|
RT |
|
Extraction Buffer P3
|
3 mL
|
15 mL
|
RT |
|
Buffer PS
|
3 mL
|
15 mL
|
RT |
|
Buffer PW(concentrate)
|
2 mL
|
10 mL
|
RT |
|
Endo-Free Buffer EB
|
2 mL
|
10 mL
|
RT |
|
RNase A
|
30 μL
|
150 μL
|
RT |
|
Endotoxin filter cartridges and collection tubes
|
10
|
50
|
RT |
|
DNA adsorption columns and collection tubes
|
10
|
50
|
RT |
Self-prepared reagents:
Isopropanol, 75% ethanol.
Notes:
- Buffer P1 with RNase A can be stored stably at 2-8°C for 6 months.
- Before the first use, add all the RNase A solution to ResuspensionBuffer P1, mix well, and store at 2–8°C. Before use, it needs to be placed at room temperature for a while and then returned to room temperature before use.
- Before first use, add anhydrous ethanol to Buffer PW according to the instructions on the reagent bottle label.
- Before use, check whether LysisBuffer P2 and Extraction Buffer E3 have crystallization or precipitation. If crystallization or precipitation occurs, place them in a 37°C water bath for a few minutes to restore clarity.
- Be careful not to directly contact LysisBuffer P2 and Extraction Buffer E3, and close the lid immediately after use.
- The quantity and purity of the extracted plasmid are related to factors such as bacterial culture concentration, strain type, plasmid size, and plasmid copy number.
Steps :
- Take 1-5 mL of bacterial culture medium and place it in a centrifuge tube. Centrifuge it at 12000 rpm for 2 min or 4000 rpm for 10 min, and discard the supernatant completely.
- Remove the centrifuge tube from the centrifuge, add 250 μL of Resuspension Buffer P1 (please check whether RNase A has been added first), and vortex to mix until the precipitate is completely suspended and the solution becomes white and turbid.
Note: If the bacterial clumps are not thoroughly mixed, the lysis effect will be affected, resulting in lower extraction volume and purity.
1. Add 250 μL Lysis Buffer P2 and invert 8-10 times.
Do not vortex to mix , and let it sit for 3-5 minutes. The solution should then become clear and viscous. (Observe the fragmentation process. If the bacterial concentration is too high, extend the lysis time to a maximum of 10 minutes.)
2. Add 250 μL of Explosion Buffer P3 and invert the tube 8-10 times until a white flocculent precipitate appears. Incubate at room temperature for 5 minutes.
3. Centrifuge at 12000 rpm for 5-10 min or 4000 rpm for 20 min at 4°C. Aspirate the supernatant and add the supernatant (about 550 μL) to the endotoxin filter column (the filter column is placed in the collection tube). Be careful to avoid any remaining foam in the supernatant.
(Excessive foam can be removed by centrifugation again), centrifuge at 12000 rpm for 1 min, and collect the filtrate in a centrifuge tube (self-prepared).
4. Add 3 times the volume of isopropanol and mix thoroughly by inverting.
5. Column equilibration: Add 200 μL of Buffer PS to the DNA adsorption column in the collection tube. Centrifuge at 12,000 rpm for 1 min. Discard the waste liquid in the collection tube and place the adsorption column back into the collection tube.
6. Transfer the mixed solution of the filtrate and isopropanol from step 6 to the equilibrated adsorption column (which has been loaded into the collection tube).
7. Centrifuge at 12,000 rpm for 1 minute, discard the waste liquid in the collection tube, and replace the adsorption column into the collection tube.
Note: The maximum volume of the adsorption column is 750 μL. If the sample volume is larger than 750 μL, it can be added in batches. 8. Add 750 μL of Buffer PW to the adsorption column (please check whether anhydrous ethanol has been added first). Centrifuge at 12,000 rpm for 1 min. Discard the waste liquid in the collection tube.
9. Place the adsorption column back into the collection tube and centrifuge at 12,000 rpm for 1 minute. Note: This step is to remove any residual ethanol in the adsorption column, as residual ethanol may affect subsequent enzymatic reactions (enzyme digestion, PCR, etc.).
10. Place the adsorption column in a new collection tube. Add 50-100 μL of Endo-Free Buffer EB to the center of the adsorption membrane. Incubate at room temperature for 2-5 minutes. Centrifuge at 12,000 rpm for 2 minutes to collect the plasmid solution in a centrifuge tube. Store the plasmid at -20°C.
Notice:
1) In order to increase the recovery efficiency of the plasmid, the obtained solution can be added back into the adsorption column and placed at room temperature for 2-5 minutes. 2 Centrifuge at 2,000 rpm min , collect the plasmid solution into a centrifuge tube .
2) For the local copy plasmid or Plasmid size >10 kb, You can Preheating Endo-Free Buffer EB in a 65-70°C water bath can increase extraction efficiency. .
