Product Introduction:
This kit uses a new silicon-based membrane technology and an optimized buffer system to allow plasmid DNA to bind to the membrane efficiently and specifically. It also uses a special buffer system and endotoxin-removing filter column to effectively remove endotoxins, proteins and other impurities.
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 Columns and Collection Tubes
|
10
|
50
|
RT |
|
DNA adsorption columns and collection tubes
|
10
|
50
|
RT |
Self-prepared reagents:
Isopropanol, 75% ethanol.
Note:
- Buffer P1 with RNase A can be stored stably for 6 months at 2-8℃.
- Before the first use, add all the RNase A solution to ResuspensionBuffer P1, mix well, and store at 2–8°C. Before use, leave it at room temperature for a while and use it after returning to room temperature.
- 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 so, place them in a 37℃ water bath for a few minutes to restore clarity.
- Be careful not to touch LysisBuffer P2 and Extraction Buffer E3 directly, 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.
Procedure :
- 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 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 is white and turbid.
Note: If the bacterial mass is not thoroughly mixed, it will affect the lysis effect, resulting in low extraction volume and purity.
1. Add 250 μL Lysis Buffer P2 and invert 8-10 times.
Do not vortex to mix , and leave it for 3-5 minutes. At this time, the solution should become clear and viscous. (The fragmentation state can be observed. If the bacterial solution concentration is too high, the lysis time can be extended to a maximum of 10 minutes).
2. Add 250 μL Express Buffer P3 and invert the tube 8-10 times until a white flocculent precipitate appears. Place the tube at room temperature for 5 min.
3. Centrifuge at 4℃ 12000 rpm for 5-10 min or 4000 rpm for 20 min, aspirate the supernatant, and add the supernatant (about 550 μL) to the endotoxin filter column (the filter column is placed in the collection tube), taking care to avoid the small amount of foam that may remain 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 supernatant to the isopropanol and mix by inverting.
5. Column equilibration: Add 200 μL 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 put the adsorption column back into the collection tube.
6. Transfer the mixed solution of filtrate and isopropanol in step 6 to the equilibrated adsorption column (loaded into the collection tube).
7. Centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back 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 Buffer PW to the adsorption column (please check whether anhydrous ethanol has been added first), centrifuge at 12,000 rpm for 1 min, and 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: The purpose of this step is to remove the residual ethanol in the adsorption column. The residual ethanol will affect the subsequent enzymatic reactions (enzyme digestion, PCR, etc.).
10. Place the adsorption column in a new collection tube, add 50-100 μL Endo-Free Buffer EB to the middle of the adsorption membrane, place at room temperature for 2-5 minutes, centrifuge at 12,000 rpm for 2 min, and collect the plasmid solution into a centrifuge tube. Store the plasmid at -20℃.
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 ,000 rpm centrifugation 2 min , collect the plasmid solution into a centrifuge tube .
2) For the local copy plasmid or Plasmid size >10 kb, Can Preheating Endo-Free Buffer EB in a 65-70℃ water bath can increase the extraction efficiency. .
