CN102242115A - Kit for extracting bacterial plasmid DNA (deoxyribonucleic acid) by magnetic beads, and extraction method thereof - Google Patents
Kit for extracting bacterial plasmid DNA (deoxyribonucleic acid) by magnetic beads, and extraction method thereof Download PDFInfo
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Abstract
The invention discloses a kit for extracting bacterial plasmid DNA (deoxyribonucleic acid) by magnetic beads, and an extraction method thereof. The kit comprises six components: a bacterial resuspension solution, a cracking solution, a neutralizing solution, a magnetic bead combining solution, a washing solution and an eluting solution. The extraction method of the kit comprises the following steps of: resuspending bacteria, cracking, neutralizing, combining with magnetic beads, washing and eluting. The plasmid DNA extracted by the invention has high purity and complete fragments, and the extraction process is convenient, quick, efficient and safe.
Description
Technical field
The invention belongs to technical field of molecular biology, particularly relate to test kit and extracting method thereof that a kind of paramagnetic particle method extracts bacteria plasmid DNA.
Background technology
Molecular biological birth is a major issue of whole natural science with development, and it makes the research of life science rise to a brand-new stage.DNA is not only basic genetic material, and plays conclusive effect in the growth of proteinic synthetic and a series of great life and mutation process.No matter be genetically engineered or protein engineering, all at first will extract and purifying DNA, particularly in genetically engineered, extremely important to the extraction of bacteria plasmid DNA.
At present, extracting the normal method that adopts of plasmid DNA from bacterium has alkaline lysis, boiling method, SDS cracking process etc., and these methods mainly are made up of the cultivation (amplification of plasmid DNA) of bacterium, the cracking (release of plasmid DNA) of bacterium and three steps of isolation and purification of plasmid DNA.Existing commercialization plasmid DNA is extracted test kit and is mostly had the imitative extractive advantage of the organic solvent of need not phenol, got well manyly compared with traditional extracting method, loaded down with trivial details high speed centrifugation, vacuum filtration, post separate and can not realize the defective that automatization is extracted with instrument but still do not break away from.
The paramagnetic particle method that grew up in recent years extracts the genomic dna technology, the genomic dna purity height of extraction, output height, and extraction step simply saves time, and makes the extraction purge process of nucleic acid become light and easy, and has obtained using widely in American-European countries.But, also do not have correlation technique and report at present about paramagnetic particle method extraction bacteria plasmid DNA.
Summary of the invention
For addressing the above problem, the purpose of this invention is to provide test kit and extracting method thereof that a kind of paramagnetic particle method extracts bacteria plasmid DNA, the plasmid DNA purity height of extraction, complete segment, leaching process is convenient and swift, highly effective and safe.
For achieving the above object, the present invention adopts following technical scheme:
A kind of paramagnetic particle method extracts the test kit of bacteria plasmid DNA, and it comprises the resuspended liquid of thalline, lysate, neutralizer, magnetic bead in conjunction with liquid, washings and six kinds of components of elutriant, the theing contents are as follows of each component:
The resuspended liquid of thalline: 40～60mmol/L glucose, PH7.6～8.4,20～30mmol/L Tris(Tutofusin tris), PH7.6～8.4,5～15mmol/L EDTA(ethylenediamine tetraacetic acid (EDTA)), 95～115 ℃ of sterilization 10～30min, 2～4 ℃ of preservations;
Lysate: 0.15～0.3mol/L NaOH, the SDS(sodium lauryl sulphate of mass concentration 0.8～1.6%);
Neutralizer: 4～5mol/L potassium acetate, 4.5～5.5mol/L glacial acetic acid;
Magnetic bead is in conjunction with liquid: 95～105ml Virahol, and 3.5～4.5ml magnetic bead suspension, the two is hybridly prepared into suspension, and wherein the magnetic bead suspension is formed according to 1:2 volume ratio mixed configuration by magnetic bead and water;
Washings: by 20～30mmol/L Tris, 8～13mmol/L EDTA, 140～160mmol/L Na salt and dehydrated alcohol mix and form, and wherein the valency volume ratio of Tris, EDTA, Na salt and dehydrated alcohol is 30%:70%;
Elutriant: 6～10mmol/L Tris, PH8.0～8.5.
Magnetic bead particle diameter in the above-mentioned magnetic bead suspension is 2～6 microns.
It is distilled water, distilled water or deionized water that above-mentioned paramagnetic particle method extracts the water that adopts in the test kit of bacteria plasmid DNA, wherein adopts distilled water in the elutriant.
Above-mentioned paramagnetic particle method extracts the test kit extracting method of bacteria plasmid DNA, and it may further comprise the steps:
Step 1, get the inoculum 1～1.5ml of ordinary method overnight incubation, join in the centrifuge tube, centrifugal 12000～15000r/min, 5～10min collects thalline, adds the resuspended liquid of 50～150 μ l thalline, 5～10 seconds of vibration, resuspended thalline;
Step 2, in resuspended thalline, add 100～150 μ l lysates, 4～8 mixings of centrifuge tube that turn upside down, as seen 2～3 minutes time length until the solution becomes thickness but limpid;
Step 3, in the split product that step 2 obtains, add 50～100 μ l neutralizers, centrifuge tube 3～7 times immediately turns upside down, floss occurring to solution precipitates, centrifugal 12000～15000 r/min, 5～10min, press close to the solution upper surface and carefully draw supernatant 150～250 μ l, transfer in another new centrifuge tube;
Step 4, in the solution that step 3 obtains, add mixing magnetic bead in conjunction with liquid 150～300 μ l, vibration mixing in 3～7 second was placed under the room temperature 8～13 minutes, centrifuge tube was placed on the magnetic force frame then, carried out magnetic and separated, and inhaled and abandoned liquid, kept magnetic bead;
Step 5, in the centrifuge tube of step 4, add 250～850 μ l washingss, vibration mixing in 3～7 second placed for 10～20 seconds, carried out magnetic with the magnetic force frame then and separated, thoroughly the exhaustion centrifuge tube loam cake and the raffinate at the pipe end were opened the dry magnetic bead of pipe lid 5～10 minutes under the room temperature (20～25 ℃);
Step 6, in the solution that step 5 obtains, add 30～150 μ l elutriants, aspirate 6～8 times mixing, placed under the room temperature 10～15 minutes or 57 ℃ of incubations 7～10 minutes, carry out magnetic then and separate, the careful supernatant liquor of drawing is transferred to new centrifuge tube, and it is standby promptly to get bacteria plasmid DNA.
In the above-mentioned step 4, at room temperature every 2～3 minutes centrifuge tube is put upside down for several times in the put procedure, with mixing solution.
In the above-mentioned step 6, at room temperature place or 57 ℃ of incubation processes in every 2～3 minutes jog centrifuge tubes 3～4 times, with mixing solution.
In the above-mentioned extraction step, resuspended liquid and elutriant are stored in 2～8 ℃, and other reagent is stored in room temperature.
When using described test kit to extract bacteria plasmid DNA, need be placed test kit in 1 hour indoor in advance, allowed it return to room temperature.Check whether lysate has precipitation,, jiggle it is mixed, build lid after taking immediately if there is precipitation 37 ℃ of temperature to bathe dissolving again in 15～25 minutes.
The yield of plasmid DNA is relevant with a plurality of factors, as the type of the plasmid copy number of cell, host cell system and substratum etc.Adopt test kit of the present invention and extracting method thereof, from the 1ml cell culture, can obtain the plasmid DNA of 1～3ug.
Because adopt aforesaid technical scheme, the present invention has following superiority:
This paramagnetic particle method extracts the test kit of bacteria plasmid DNA, the plasmid DNA purity height of its extraction, and complete segment, for low copy plasmid, the purifying ability is powerful, and is cheap, economical and practical; The extracting method that this paramagnetic particle method extracts bacteria plasmid DNA does not use toxic reagent, outside the bacteria-removing liquid conventional processing, the paramagnetic particle method extraction need not centrifugal, do not need vacuum filtration, post separation etc. yet, except that can manual extraction the operation, also be suitable for the automation equipment operation especially, the purity of plasmid DNA is 1.8 ± 0.1, and extraction time is short, environment friendly and pollution-free.
Description of drawings
Fig. 1 is the agarose electrophoresis figure of the plasmid DNA of test kit extraction of the present invention; Wherein 1,2 swimming lanes are that commercially available centrifugal method plasmid DNA is extracted the plasmid DNA band that test kit extracts, and 3,4 swimming lanes are the plasmid DNA band that test kit of the present invention extracts.
Embodiment
As can be seen from Figure 1, the plasmid DNA that test kit of the present invention extracts aspect purity and complete segment, with the general goods plasmid DNA extract test kit compare not poor about.
Can be described in further detail the present invention with reference to following examples; But following examples only are illustrations, and the present invention is not limited to these embodiment.
Embodiment one
A kind of paramagnetic particle method extracts the test kit extracting method of bacteria plasmid DNA, and it specifically may further comprise the steps:
Step 1, get the inoculum of 1.0ml overnight incubation, join in the centrifuge tube (EP) of 1.5ml centrifugal 7 minutes of 12000 r/min, abandon supernatant, keep thalline, add the resuspended liquid of 50 μ l, eddy oscillating 5 seconds, resuspended thalline, guaranteeing does not have the visible fritter after resuspended;
Step 2, add 100 μ l lysates in resuspended thalline, 5 mixings of centrifuge tube that turn upside down continue 2 minutes, and as seen high vibration not, otherwise can cause genomic dna to be sheared up to the solution becomes thickness but limpid;
Step 3, in the split product that step 2 obtains, add 75 μ l neutralizers, centrifuge tube 5 times immediately turns upside down, floss occurring to solution precipitates, centrifugal 10 minutes of 12000 r/min, form white precipitate closely, press close to the solution upper surface and carefully draw supernatant 200 μ l, transfer in another new centrifuge tube; Note not sucking throw out, otherwise can cause genomic dna to pollute; ,
Step 4, combination: in the solution that step 3 obtains, add the magnetic bead of mixing in conjunction with liquid 250 μ l, vibrate 5 second mixing, placed 10 minutes under the room temperature, wherein centrifuge tube is put upside down mixing 3 times every 3 minutes, after 10 minutes, centrifuge tube is placed on the magnetic force frame, carries out magnetic and separate, abandon liquid with thin suction nozzle suction, keep magnetic bead;
Step 5, washing: in the centrifuge tube of step 4, add 500 μ l washingss, vibrate 5 second mixing, placed for 15 seconds, carry out magnetic with the magnetic force frame then and separate, thoroughly the exhaustion centrifuge tube loam cake and the raffinate at the pipe end were opened the dry magnetic bead of pipe lid 5 minutes under the room temperature (20～25 ℃);
Step 6, wash-out: in the solution that step 5 obtains, add 60 μ l elutriants, aspirate 6 times mixing, placed 10 minutes under the room temperature, every 3 times mixings of 3 minutes jog centrifuge tubes, carry out magnetic then and separate, carefully draw supernatant liquor and be transferred to new centrifuge tube, it is standby promptly to get bacteria plasmid DNA.
Embodiment two
A kind of paramagnetic particle method extracts the test kit extracting method of bacteria plasmid DNA, and it specifically may further comprise the steps:
Step 1, get the inoculum of 1.5ml overnight incubation, join in the centrifuge tube (EP) of 2.0ml centrifugal 10 minutes of 13000 r/min, abandon supernatant, keep thalline, add the resuspended liquid of 65 μ l, eddy oscillating 6 seconds, resuspended thalline, guaranteeing does not have the visible fritter after resuspended;
Step 2, add 130 μ l lysates in resuspended thalline, 5 mixings of centrifuge tube that turn upside down continue 3 minutes, and as seen high vibration not, otherwise can cause genomic dna to be sheared up to the solution becomes thickness but limpid;
Step 3, in the split product that step 2 obtains, add 100 μ l neutralizers, centrifuge tube 5 times immediately turns upside down, floss occurring to solution precipitates, centrifugal 10 minutes of 13000 r/min, form white precipitate closely, press close to the solution upper surface and carefully draw supernatant 230 μ l, transfer in another new centrifuge tube; Note not sucking throw out, otherwise can cause genomic dna to pollute; ,
Step 4, combination: in the solution that step 3 obtains, add the magnetic bead of mixing in conjunction with liquid 260 μ l, vibrate 5 second mixing, placed 10 minutes under the room temperature, wherein centrifuge tube is put upside down mixing 3 times every 3 minutes, after 10 minutes, centrifuge tube is placed on the magnetic force frame, carries out magnetic and separate, abandon liquid with thin suction nozzle suction, keep magnetic bead;
Step 5, washing: in the centrifuge tube of step 4, add 700 μ l washingss, vibrate 5 second mixing, placed for 10 seconds, carry out magnetic with the magnetic force frame then and separate, thoroughly the exhaustion centrifuge tube loam cake and the raffinate at the pipe end were opened the dry magnetic bead of pipe lid 10 minutes under the room temperature (20～25 ℃);
Step 6, wash-out: in the solution that step 5 obtains, add 100 μ l elutriants, aspirate 6 times mixing, 57 ℃ of incubations 10 minutes, every 3 times mixings of 3 minutes jog centrifuge tubes, carry out magnetic then and separate, carefully draw supernatant liquor and be transferred to new centrifuge tube, it is standby promptly to get bacteria plasmid DNA.
Claims (7)
1. a paramagnetic particle method extracts the test kit of bacteria plasmid DNA, and it is characterized in that: it comprises the resuspended liquid of thalline, lysate, neutralizer, magnetic bead in conjunction with liquid, washings and six kinds of components of elutriant, the theing contents are as follows of each component:
The resuspended liquid of thalline: 40～60mmol/L glucose, PH7.6～8.4,20～30mmol/L Tris, PH7.6～8.4,5～15mmol/L EDTA, 95～115 ℃ of sterilization 10～30min, 2～4 ℃ of preservations;
Lysate: 0.15～0.3mol/L NaOH, the SDS of mass concentration 0.8～1.6%;
Neutralizer: 4～5mol/L potassium acetate, 4.5～5.5mol/L glacial acetic acid;
Magnetic bead is in conjunction with liquid: 95～105ml Virahol, and 3.5～4.5ml magnetic bead suspension, the two is hybridly prepared into suspension, and wherein the magnetic bead suspension is formed according to 1:2 volume ratio mixed configuration by magnetic bead and water;
Washings: by 20～30mmol/L Tris, 8～13mmol/L EDTA, 140～160mmol/L Na salt and dehydrated alcohol mix and form, and wherein the valency volume ratio of Tris, EDTA, Na salt and dehydrated alcohol is 30%:70%;
Elutriant: 6～10mmol/L Tris, PH8.0～8.5.
2. paramagnetic particle method according to claim 1 extracts the test kit of bacteria plasmid DNA, and it is characterized in that: the magnetic bead particle diameter in the described magnetic bead suspension is 2～6 microns.
3. paramagnetic particle method according to claim 1 extracts the test kit of bacteria plasmid DNA, and it is characterized in that: the water that adopts in described each component is distilled water, distilled water or deionized water, wherein adopts distilled water in the elutriant.
4. described test kit extracting method of claim 1, it is characterized in that: it may further comprise the steps:
Step 1, get the inoculum 1～1.5ml of ordinary method overnight incubation, join in the centrifuge tube, centrifugal 12000～15000r/min, 5～10min collects thalline, adds the resuspended liquid of 50～150 μ l thalline, 5～10 seconds of vibration, resuspended thalline;
Step 2, in resuspended thalline, add 100～150 μ l lysates, 4～8 mixings of centrifuge tube that turn upside down, as seen 2～3 minutes time length until the solution becomes thickness but limpid;
Step 3, in the split product that step 2 obtains, add 50～100 μ l neutralizers, centrifuge tube 3～7 times immediately turns upside down, floss occurring to solution precipitates, centrifugal 12000～15000 r/min, 5～10min, press close to the solution upper surface and carefully draw supernatant 150～250 μ l, transfer in another new centrifuge tube;
Step 4, in the solution that step 3 obtains, add mixing magnetic bead in conjunction with liquid 150～300 μ l, vibration mixing in 3～7 second was placed under the room temperature 8～13 minutes, centrifuge tube was placed on the magnetic force frame then, carried out magnetic and separated, and inhaled and abandoned liquid, kept magnetic bead;
Step 5, in the centrifuge tube of step 4, add 250～850 μ l washingss, vibration mixing in 3～7 second placed for 10～20 seconds, carried out magnetic with the magnetic force frame then and separated, thoroughly the exhaustion centrifuge tube loam cake and the raffinate at the pipe end were opened the dry magnetic bead of pipe lid 5～10 minutes under the room temperature;
Step 6, in the solution that step 5 obtains, add 30～150 μ l elutriants, aspirate 6～8 times mixing, placed under the room temperature 10～15 minutes or 57 ℃ of incubations 7～10 minutes, carry out magnetic then and separate, the careful supernatant liquor of drawing is transferred to new centrifuge tube, and it is standby promptly to get bacteria plasmid DNA.
5. extracting method according to claim 4 is characterized in that: in the step 4, at room temperature every 2～3 minutes centrifuge tube is put upside down for several times in the put procedure.
6. extracting method according to claim 4 is characterized in that: in the step 6, at room temperature place or 57 ℃ of incubation processes in every 2～3 minutes jog centrifuge tubes 3～4 times.
7. extracting method according to claim 4 is characterized in that: described resuspended liquid and elutriant are stored in 2～8 ℃, and other reagent is stored in room temperature.
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| CN113388610B (en) * | 2021-06-28 | 2022-07-05 | 武汉纺织大学 | Kit for rapidly extracting bacterial plasmid DNA by paramagnetic particle method and extraction method |
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| CN113846088B (en) * | 2021-09-07 | 2024-03-12 | 武汉奥科鼎盛生物科技有限公司 | Kit and method for extracting plasmid DNA |
| CN114107288A (en) * | 2021-12-22 | 2022-03-01 | 艾柏森(江苏)生物科技有限公司 | Kit for extracting plasmid DNA by paramagnetic particle method and method for extracting plasmid DNA |
| CN114395553A (en) * | 2021-12-23 | 2022-04-26 | 北京擎科生物科技有限公司 | Magnetic bead method plasmid extraction kit, preparation method and plasmid extraction method |
| CN114657174A (en) * | 2022-03-29 | 2022-06-24 | 湖南科技学院 | Kit for extracting bacterial plasmid by alkaline cracking method and method thereof |
| CN114657174B (en) * | 2022-03-29 | 2023-07-25 | 湖南科技学院 | Kit for extracting bacterial plasmid by alkaline lysis method and method thereof |
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