CommaPrepTM Plasmid Large Purification Column Instructions
This manual (version 1.0) is for products numbered DNAK1003 and all technical literature is available on the company's website at Please visit the company website to verify that the technical manual you are using is the latest version.
product description
The CommaPrepTM range uses classic silica membrane adsorption technology. The plasmid purification column is composed of a collection tube, an adsorption column, a sieve plate, a silica gel membrane and a pressure ring. The principle of silica gel membrane adsorption technology is that silica gel membrane specifically adsorbs DNA under high salt and low pH state; while in low salt or aqueous solution, DNA is eluted. The method has the characteristics of simple, non-toxic, high recovery rate and good purification effect. The plasmid DNA extracted by the plasmid extraction column can be applied to various conventional operations, including enzyme digestion, PCR, sequencing, ligation, transformation and transfection of various cells.
Features
The quality of the silica gel membrane is stable. In a specific solution environment, the adsorption capacity of nucleic acid is strong, the environment is changed, the nucleic acid is easily eluted, the difference between the column and the column is extremely small, and the repeatability is good;
The sieve plate is made of special materials and can withstand high speed centrifugation;
The sieve plate is smaller than the area, the static electricity is small, and the DNA adsorption is extremely low;
Fully automated assembly, reducing human factors;
The bottom of the adsorption column is designed with a Luer interface to facilitate centrifugation.
Scope of application
Large amount of plasmid was extracted and purified.
Product parameters and specifications
Nucleic acid adsorption amount: 0-500μg
Collection tube: 50mL centrifuge tube adsorption column diameter: 24.0mm
High speed centrifugal membrane pore size: 50μm
High speed centrifugal film thickness: 0.3-0.5mm
Silicone membrane pore size: 1μm
Steps
A. Column equilibration Add 2.5 mL of equilibration solution to the adsorption column placed in a 50 mL collection tube, centrifuge at 8000 rpm for 2 min, drain the waste liquid from the collection tube, and return the adsorption column to the collection tube.
B. Bacterial lysis
1. Take 25mL of bacterial solution, centrifuge at 12000rpm for 1-2min, pour the supernatant as much as possible, and collect the cells.
Note: 25mL-100mL is recommended for high copy and 50mL-200mL for low copy.
If the collected bacterial liquid is large, after centrifuging to remove the supernatant, add the bacterial liquid in the same 50 mL tube, repeat this step, and collect the bacterial liquid into a collection tube.
2. Resuspend the bacterial pellet with 10 mL of solution P1 (previously added to RNaseA), and use a pipette or vortex shaker to completely suspend the bacterial cell pellet. If there is a mixture that is not thoroughly mixed, it will affect the lysis, resulting in the amount of extraction. And the purity is low.
Note: If it is a low-copy plasmid, colleagues who increase the amount of bacteria will increase the amount of P1, P2, and P3 proportionally.
3. Add 10 mL of solution P2, gently invert 4-6 times, and let stand for 5 minutes at room temperature (do not exceed 5 minutes). At this time, the bacterial liquid becomes clear and viscous. If it is turbid, it may be due to excessive bacterial cells, and the lysis is not complete, reducing the amount of bacteria.
4. Add 10mL solution P3, immediately gently flip it up and down 4-6 times, mix well and appear white flocculent precipitate, leave it at room temperature for 5min. Centrifuge at 12000 rpm for 10 min to precipitate white to the bottom of the tube.
Note: If the supernatant is not clear enough, you will need to continue centrifugation.
5. Carefully pipette the supernatant into a new centrifuge tube to avoid drawing a floating white precipitate. Or carefully pour all the solution into the filter (to avoid sedimentation), slowly push the pusher to filter, and collect the filtrate in a clean centrifuge tube.
C. Extraction and purification of plasmid DNA
1. Transfer the supernatant obtained in the previous step (no more than 25mL each time) into the adsorption column (the adsorption column is placed in the collection tube), centrifuge at 12000rpm for 1min, drain the waste liquid from the collection tube, and put the adsorption column back into the collection. In the tube.
2. Add 10 mL of rinse solution (previously added with absolute ethanol at 1:3), centrifuge at 12000 rpm for 1 min, discard the waste solution, and return the column to the collection tube.
3. Repeat step 2 once.
4. Centrifuge at the highest speed for 5 min, dry the residual rinse, and use the tip to remove any rinsing liquid that may remain between the pressure ring and the column wall. Open the lid of the column and leave it at room temperature for a few minutes to thoroughly dry the rinse in the adsorbent.
5. Place the adsorption column in a new 50 mL centrifuge tube, add 2.5 mL of eluate to the middle of the adsorption membrane, leave it at room temperature for 5 min, centrifuge at 12000 rpm for 2-5 min. If the efficiency of the recovery of the plasmid is increased, the obtained solution can be re-added to the adsorption column, and this step is repeated. After completion, the DNA adsorption column can be discarded and the purified plasmid stored at -20 °C.
Detection of plasmid DNA concentration
The resulting plasmid DNA can be detected for concentration and purity by agarose gel electrophoresis and ultraviolet spectrophotometry. Purified plasmid DNA OD260/OD280, usually around 1.8-2.0.
Precautions
1. All centrifugation steps were completed at room temperature unless otherwise stated.
2. Balance the adsorption column before use to maximize the activation of the silica gel membrane and increase the yield. And the balanced column is best used immediately, and the long time will affect the use effect.
3. The pH of the solution before the upper column should be less than 7.
4. The concentration of ethanol in the rinsing process is not less than 50%, generally between 55% and 80%.
5. Finally eluting, ddH2O or TE (10 mmol/L Tris-Cl, 1 mmol/L EDTA (pH 8.0)) can be used. If you are storing DNA for a long time, it is recommended to use TE.
6. If ddH2O is used as the eluent, ensure that the pH is between 7.5 and 8.0. A pH below 7.0 will reduce the elution efficiency.
7. The volume of the eluent is not less than 1 mL.
Solution preparation
Solution P1: 50 mM Tris-HCl (pH 7.5), 10 mM EDTA, 100 μg/ml RNase A
Solution P2: 0.2M NaOH, 1% SDS
Solution P3: 4.09 M guanidine hydrochloride, 0.759 M potassium acetate, 2.12 M glacial acetic acid, pH about 4.2.
Equilibrate: 750 mM NaCl, 50 mM MOPS, pH 7.0, 15% isopropanol (V/V), 0.15% Triton X-100 (V/V)
Rinsing solution: 60 mM potassium acetate, 10 mM Tris-HCl (pH 7.5), 60% ethanol eluent: 1.25 M NaCl, 50 mM Tris-HCl pH 8.5, 15% isopropanol
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