CN109652409A - A method of extracting aureus plasmid - Google Patents
A method of extracting aureus plasmid Download PDFInfo
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- CN109652409A CN109652409A CN201910153632.4A CN201910153632A CN109652409A CN 109652409 A CN109652409 A CN 109652409A CN 201910153632 A CN201910153632 A CN 201910153632A CN 109652409 A CN109652409 A CN 109652409A
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Abstract
The invention discloses a kind of methods for extracting aureus plasmid.This method comprises the following steps: first using reagent, lysozyme and the staphylococcus enzymatic lysis microorganism of cracking microorganism, and carries out physical wall breaking using glass powder, cracks staphylococcus aureus thallus sufficiently, obtain cellular lysate liquid；Then the reagent for being used for precipitating proteins and genomic DNA is added into the cellular lysate liquid, obtains the mixed liquor precipitated containing protein precipitation and genomic DNA；It is centrifuged at a high speed, supernatant is taken to be transferred in adsorption column, carry out absorption centrifugation, be enriched with Plasmid DNA；Rinsing liquid is added and carries out desalination centrifugation, ultrapure water elution centrifugation is added, obtains Plasmid DNA.The broken wall efficiency that method of the invention uses physics and chemical method to improve staphylococcus aureus simultaneously, save the cost improve the rate of recovery of plasmid in plasmid extraction process.
Description
Technical field
The present invention relates to molecular biology fields, more particularly to a kind of method for extracting aureus plasmid.
Background technique
Plasmid is that the hereditary unit independently replicated is able to carry out outside chromosome.Generally comprise supercoil, open loop and linear three
Kind of structure is present in the biology such as many bacteriums and saccharomycete based on cyclic configuration or even the mitochondria of plant etc. is thin
In born of the same parents' device.The length of plasmid is generally in 2-20kbp, and molecular weight is generally 106 or more.
Plasmid DNA is the most common carrier of genetic engineering.Some plasmids contain certain drug resistant gene, have some plasmids to take
The gene of band can assign cell additional physiological metabolism, or even its pathogenicity is improved in some bacteriums.It can
A useful target DNA fragment by recombinant DNA technology, is sent into recipient cell and goes to be bred and be expressed, and one
As for, the presence or absence of plasmid survives no conclusive effect to host cell.Therefore the quality that plasmid extracts is to subsequent
Molecular biology experiment achievement whether play a key role.
Currently, the plasmid extraction kit being commercially available on the market is mainly both for the pumping of plasmid in Gram-negative bacteria
It mentions, for the extracting of plasmid in gram-positive bacteria especially staphylococcus aureus, there is no dedicated kits.But it is golden
Staphylococcus aureus can cause many serious infection as important pathogen, can be to dynamic if wherein there is r plasmid
The object even mankind cause significant damage.
Summary of the invention
In order to overcome the disadvantages mentioned above and deficiency of the prior art, it is an object of the invention to propose one kind to golden yellow grape
The method that coccus plasmid extracts, based on breaking walls and cracking method chemical in existing plasmid extraction kit, by chemistry and object
Reason cleavage method combines, and extracts process to entire Staphylococcus aureus plasmid and optimize, and reaches and is based on now on the market
Existing plasmid extraction kit can successfully come out the plasmid extraction in staphylococcus aureus, for further studying.
The purpose of the present invention is achieved through the following technical solutions.
A kind of plasmid extracting method of staphylococcus aureus provided by the invention, includes the following steps:
(1) by the S. aureus Inoculate of the plasmid containing purpose in brain heart oxoid (BHI)/Antibiotic medium, shaking table
Culture expands bacterium solution, obtains primary bacterium solution；
(2) primary bacterium solution described in inoculation step (1) connects into 100-150mL brain heart oxoid (BHI)/Antibiotic medium
Kind amount is 1-3mL, and shaking table culture expands bacterium solution, obtains secondary bacterium solution；
(3) secondary bacterium solution described in 60mL-100mL step (2) is subjected to room temperature centrifuge separation；Fall to abandon upper layer culture after centrifugation
Liquid obtains the centrifuge tube equipped with staphylococcus aureus precipitating centrifuge tube left-hand thread in exhausting raffinate on blotting paper；
(4) lysozyme and staphylococcus lysozyme are added in the reagent of cracking microorganism；Obtain cracking Staphylococcus aureus
The mixed liquor of bacterium；
(5) mixed liquor of step (4) the cracking staphylococcus aureus is added to described in step (3) equipped with golden yellow grape
In the centrifuge tube of coccus precipitating, be vortexed be resuspended processing to thallus be all resuspended, in centrifuge tube without precipitating until, then at constant temperature bath
Reason, obtains re-suspension liquid；
(6) re-suspension liquid described in 500-520 microlitres of step (5) is transferred in another centrifuge tube, glass powder is added, be vortexed mixed
It is even to obtain the re-suspension liquid containing glass powder；
(7) alkaline lysis liquid is added in the re-suspension liquid containing glass powder described in step (6), covers centrifuge tube lid, overturn centrifuge tube
5-10 times, obtain mixed liquor；
(8) Proteinase K is added in step (7) described mixed liquor, covers centrifuge tube lid, reverse centrifuge tube 5-10 times, so
After be stored at room temperature 10-15 minutes, be mixed by inversion 5-10 times every 2-3 minutes therebetween and obtain the mixed liquor containing Proteinase K；
(9) 420-450 microlitres of rapid precipitation buffer is added in the mixed liquor containing Proteinase K described in step (8), cover from
The lid of heart pipe overturns 10-15 times, obtains suspension；
(10) suspension for obtaining step (9) carries out room temperature centrifuge separation, obtains supernatant；
(11) adsorption column is placed in collecting pipe respectively, supernatant obtained by step (10) is added in adsorption column respectively, room temperature from
Heart separation abandons the filtrate in collecting pipe, adsorption column is recovered in collecting pipe；
(12) by rinsing liquid 1 in adsorption column, room temperature stands 3-5 minutes, and room temperature centrifuge separation abandons filtrate, absorption column sleeve
It recycles in collector；
(13) by rinsing liquid 2 in adsorption column, room temperature centrifuge separation abandons filtrate, adsorption column is recovered in collecting pipe；
(14) step (13) are repeated；
(15) room temperature centrifugation step (14) resulting adsorption column；
It (16) is in 1.5mL centrifuge tube, by elution buffer or sterilizing in sterilized volume by the absorption column sleeve after centrifugation
The film center of adsorption column is added in water, and room temperature centrifugal treating after standing discards adsorption column, in the centrifuge tube that volume is 1.5mL
To eluent, the purpose plasmid is contained in eluent；
(17) step (16) described eluent is transferred in another adsorption column of step (15) and is eluted, room temperature after standing
Centrifugal treating discards adsorption column, and secondary eluent is obtained in the centrifuge tube that volume is 1.5mL, is contained in secondary eluent dense
Spend the purpose plasmid increased；
(18) and so on until the elution of all adsorption columns completely, obtains final eluent, that is, is enriched with the elution of purpose Plasmid DNA
Liquid (eluent of the Plasmid DNA of purpose containing high-purity)；
(19) eluent (eluent containing high-purity plasmid DNA) that purpose Plasmid DNA is enriched with described in step (18) is saved
It is stored refrigerated in refrigerator.
Further, purpose plasmid described in step (1) is wild plasmid, and the purpose plasmid contains anti-antibiotic
Gene；The temperature of the shaking table culture is 37 DEG C, and the time of culture is 10-12 hours.
Further, purpose plasmid described in step (1) is wild plasmid, and the purpose plasmid contains anti-antibiotic
Gene；The temperature of shaking table culture described in step (1) is 37 DEG C, and the time of shaking table culture is 10-12 hours；In step (2)
The dosage of the primary bacterium solution is 1-3mL, i.e., inoculum concentration is 1-3mL；Brain heart oxoid described in step (2)/antibiotic culture
The dosage of base is 100-150mL；The temperature of shaking table culture described in step (2) is 37 DEG C, and incubation time is 12-16 hours.
Further, the volume of secondary bacterium solution described in step (3) is 60mL-100mL；Room temperature described in step (3) from
The rate of heart separation is 6000-8000 revs/min, and the time of centrifuge separation is 10-15 minutes.
Further, the additional amount of lysozyme described in step (4) is to add in every milliliter of reagent for cracking microorganism
5-8mg；The additional amount of the staphylococcus lysozyme is to add 1-3mg in every milliliter of reagent for cracking microorganism.
Further, the additional amount that the mixed liquor of staphylococcus aureus is solved described in step (5) is 3-5mL；Step (5)
Described in constant temperature bath processing temperature be 37 degrees Celsius；The time of constant temperature bath processing is 20-30 minutes；
Further, the dosage of re-suspension liquid described in step (6) is 500-520 microlitres；The partial size of glass powder described in step (6)
For 0.1-0.6mm, for the glass powder using high-temperature sterilization is preceding needed, the additional amount of the glass powder is 180-200mg；Step (6)
Described in the be vortexed vortex time of mixing be 8-10 minute, the vortex rate of the mixing that is vortexed is 2600-2800 revs/min；Step
(7) additional amount of alkaline lysis liquid described in is 500-520 microlitres.
Further, Proteinase K described in step (8) is a kind of strength protein dissolution separated from Candida albicans
Enzyme；The concentration of Proteinase K is 20-25mg/mL, and the additional amount of Proteinase K is 10-12 microlitres.
Further, the additional amount of rapid precipitation buffer described in step (9) is 420-450 microlitres；In step (10)
The rate of the room temperature centrifuge separation is 12000-13000 revs/min, and the time of room temperature centrifuge separation is 10-15 minutes；Step
(11) rate of the centrifuge separation of room temperature described in is 8000-10000 revs/min, and the time of room temperature centrifuge separation is 30-60 seconds.
Further, step (11) adsorption column and collecting pipe are one-to-one quantitative relations, provided by the invention to mention
Take method preferably 6 adsorption columns and 6 collecting pipes.
Further, the additional amount of rinsing liquid 1 described in step (12) is 600-630 microlitres；Described in step (12) often
The time that temperature is stood is 3-5 minutes；The rate of the centrifuge separation of room temperature described in step (12) is 8000-10000 revs/min, often
The time of temperature centrifuge separation is 30-60 seconds；The additional amount of rinsing liquid 2 described in step (13) is 620-650 microlitres；Step (13)
Described in room temperature centrifuge separation rate be 8000-10000 revs/min, room temperature centrifuge separation time be 30-60 seconds.
Further, the centrifugal rotational speed of the centrifugation of room temperature described in step (15) is 12000-13000 revs/min, centrifugation
Time is 3-5 minutes；The additional amount of elution buffer described in step (16) or aqua sterilisa is 60-100 microlitres；Step (16)
Time with step (17) described standing is 1-3 minutes；Step (16) and the rate of step (17) the room temperature centrifugal treating are
12000-13000 revs/min, the time of centrifugation is 1-2 minutes；Stored refrigerated temperature is preferably 20 described in step (19)
℃。
Further, alkaline lysis liquid, step described in the reagent of microorganism, step (7) are cracked described in step (4)
(9) rapid precipitation buffer described in, adsorption column described in step (11), rinsing liquid 1 described in step (12), in step (13)
Elution buffer described in the rinsing liquid 2 and step (16) derives from plasmid extraction kit.
Further, cracking microorganism of the present invention uses chemical reagent cracking and mutually ties with glass powder physical disruption
The method of conjunction obtains the complete staphylococcus aureus lysate of broken wall.
Further, it needs that Proteinase K is added after the alkaline lysis, memebrane protein is made to degrade, DNA is sufficiently free.
Further, method provided by the invention elutes multiple adsorption columns using elution buffer or aqua sterilisa repeatedly, obtains
Obtain the Plasmid DNA of high-purity.
The plasmid that extracting method of the invention is extracted contains anti-antibiotic resistance gene, this is the present invention to existing extracting method
Optimization, on the one hand the anti-antibiotic resistance gene can be used as the label of purpose plasmid, on the other hand the anti-antibiotic resistance gene
Us can be helped to screen out staphylococcus aureus.It is understood that extracted plasmid size, so that we are in late detection
In can quickly detect purpose plasmid, save experimental period, extracting method provided by the invention can be verified in the short time
Effect；Secondly, anti-antibiotic resistance gene can help staphylococcus aureus to survive in antibiotic culture medium, and
Miscellaneous bacteria without anti-antibiotic resistance gene can not survive, this avoid occur in experimentation of the present invention living contaminants cause into
The case where degree is slowly or result is interfered, improve the accuracy of extracting method provided by the invention.Therefore, described anti-anti-
Raw plain gene is a tool in extracting method provided by the invention, as long as ensuring to extract object is Staphylococcus aureus
Bacterium, extracting method provided by the invention are also applied for extracting without anti-antibiotic resistance gene plasmid.
Extracting method provided by the invention is collection matter as much as possible on the basis of considering adsorption column bearing capacity
Grain DNA, plasmid extracting concentration is improved, therefore 6 adsorption columns has been selected to work at the same time, this is the present invention to the excellent of the prior art
Change.
Compared with prior art, the invention has the advantages that and the utility model has the advantages that
(1) method that a kind of pair of aureus plasmid proposed by the present invention extracts uses chemical reagent and cracks
The method combined with glass powder physical disruption allows aureus cell wall to crack more complete, reduces molten
The dosage of bacterium enzyme and molten Portugal bacterium enzyme to save experimental cost, and can significantly shorten experimental period.Therefore make gold
The extraction process of the ribonucleic acid of staphylococcus aureus plasmid has the characteristics that efficient, quick, succinct.
(2) proposed by the present invention that Proteinase K is added during extracting aureus plasmid, memebrane protein drops
Solution, DNA is sufficiently free, improves product stability；Institute's upgrading grain ribonucleic acid is applicable to various routine operations, including digestion,
PCR, sequencing, connection, conversion, library screening, In Vitro Translation, some conventional passage cells of transfection etc..
(3) extracting method provided by the invention is receipts as much as possible on the basis of considering adsorption column bearing capacity
Collect Plasmid DNA, improve plasmid extracting concentration, can preferably 6 adsorption columns work at the same time, improve extraction efficiency, reduce and mention
The time is taken, is that the present invention advanced optimizes the prior art.
Detailed description of the invention
Fig. 1 be the embodiment of the present invention 1 extracted from 80 milliliters of staphylococcus aureus culture solutions obtain plasmid electrophoresis inspection
Mapping.
Specific embodiment
Implementation of the invention is described further below in conjunction with the drawings and specific embodiments, but embodiment party of the invention
Formula is without being limited thereto.If being that those skilled in the art can it is noted that having the process or parameter of not special detailed description below
Referring to the prior art understand or realize.
Embodiment 1
The Plasmid DNA Trace bio-element kit (HiPure Plasmid Micro kit C) produced using Magen company, goods
Number: P1001-02C, concrete composition: RNase A, Buffer P1 Plus, Buffer P2 Plus, Buffer P3, Buffer
PW1、Buffer PW2、Buffer TE、HiPure DNA Mini Column Ⅱ、2 ml Collection Tube。
(1) will contain the S. aureus Inoculate of purpose plasmid (the purpose plasmid contain anti-kanamycin gene) in
5ml contains in the BHI culture medium of 15 μ g/ml kanamycins (KAN), 37 DEG C of shaking table culture 10 hours a small amount of amplification bacterium solutions；
(2) it is added in 250ml culture bottle in BHI broth of the 100ml containing 15 μ g/ml KAN, is inoculated with the first of 2ml step (1)
Grade bacterium solution is into culture bottle, 37 DEG C of shaking table cultures, 12 hours amplification bacterium solutions；
(3) the staphylococcus aureus bacterium solution for collecting 80ml step (2) carries out room temperature centrifuge separation, and the revolving speed of centrifuge separation is
6000 revs/min, the time of centrifuge separation is 10 minutes, abandons culture supernatants, and centrifuge tube left-hand thread is residual in blotting paper exhaustion
Liquid；
(4) take the reagent Buffer P1 Plus/RNase A of 3ml cracking microorganism in containing lysozyme and staphylococcus enzyme
5ml centrifuge tube in, make the concentration 5mg/ml of lysozyme, the concentration of staphylococcus enzyme is 1mg/ml(lysozyme and molten grape
Coccus enzyme is bought in Keyinbio company, and article No. is respectively 12650-88-3 and 9011-93-2；).
(5) mixed liquor of 3ml step (4) the cracking staphylococcus aureus is taken to be added to step (3) described equipped with gold
In the centrifuge tube of staphylococcus aureus precipitating, being vortexed to be resuspended to handle to thallus is all resuspended, without precipitating, 37 DEG C of warm bath in centrifuge tube
20 minutes；
(6) 500 microlitres of step (5) re-suspension liquids are shifted respectively into new 2mL centrifuge tube, are separately added into 200mg glass powder (partial size
For 0.1-0.6mm, preceding high-temperature sterilization is used), it 2600 revs/min, is vortexed and mixes 8 minutes, vortex mixer input power is
1.2W, output power 0.8W are completely severed the cell wall of staphylococcus aureus；
(7) 500 microlitres of alkaline lysis liquid Buffer P2 Plus are added in each centrifuge tube in step (6), overturn centrifuge tube 5
It is secondary, make obtained cellular lysate in step (6)；
(8) 10 microlitres of 20mg/ml Proteinase Ks (purchase is in Merck KGaA company, article No.: 39450-01-6) to step (7) is taken
It in resulting lysate, is mixed by inversion centrifuge tube 5 times, is stored at room temperature 10 minutes, is mixed by inversion 6 times and obtains at intervals of two minutes therebetween
Mixed liquor containing Proteinase K；
(9) it takes 420 microlitres of rapid precipitation buffer solution B uffer P3 into step (8) resulting lysate, is mixed by inversion 10 times and allows
Solution thoroughly neutralizes；
(10) suspension obtained by step (9) is subjected to room temperature centrifuge separation, the revolving speed of centrifuge separation is 13000 revs/min, centrifugation
The isolated time is 10 minutes；
(11) 6 adsorption columns (adsorption column used in embodiment 1 is HiPure DNA Mini Column II) are placed in 6
In collecting pipe (2 ml Collection Tube), respectively transfer step (10) resulting supernatant in adsorption column, room temperature from
Heart separation, the revolving speed of centrifuge separation are 8000 revs/min, and the time of centrifuge separation is 30 seconds, abandon filtrate, adsorption column is recovered
In collecting pipe；
(12) it takes 600 microlitres of rinsing liquids, 1 Buffer PW1 in the adsorption column of step (11), stands 3 minutes, room temperature centrifugation point
From the revolving speed of centrifuge separation is 8000 revs/min, and the time of centrifuge separation is 30 seconds, abandons filtrate, adsorption column is recovered collection
Guan Zhong；
(13) take 620 microlitres of rinsing liquids, 2 Buffer PW2 in the adsorption column of step (12), room temperature centrifuge separation, centrifuge separation
Revolving speed be 8000 revs/min, time of centrifuge separation is 30 seconds, abandons filtrate, adsorption column is recovered in collecting pipe；
(14) step (13) are repeated；
(15) room temperature step with centrifugal separation (14) resulting adsorption column, the revolving speed of centrifuge separation are 12000 revs/min, centrifugation point
From time be 3 minutes, dry adsorption column removes ethyl alcohol；
(16) an absorption column sleeve in step (15) is taken 60 microlitres of elution buffers in the centrifuge tube of the 1.5ml of sterilizing
Buffer TE to adsorption column film center, stand 1 minute, room temperature be centrifuged eluted dna, be centrifuged elution revolving speed be 12000 turns/
Minute, the time of centrifuge separation is 1 minute；
(17) product that step (16) elute is transferred in another adsorption column of step (15) and is eluted, operating procedure with
Step (16) is identical；
(18) and so on until the elution of all adsorption columns completely, obtains high-purity plasmid DNA；
(19) adsorption column in (18) is discarded, (18) resulting eluent is stored in -20 DEG C.
(20) it detects: using mass percent concentration to carry out for 0.8% Ago-Gel the Plasmid DNA product of elution
Detection, buffer are that (TAE buffer is made of TAE buffer trishydroxymethylaminomethane, acetic acid and ethylenediamine tetra-acetic acid
Buffer), voltage be 1000V under conditions of electrophoresis 90min, as a result see shown in Fig. 1, extract aureus plasmid
DNA as a result, electrophoretic band from left to right successively are as follows: Plasmid DNA (English name of plasmid be plasmid), marker
Marker(is bought from Bo Maide Bioisystech Co., Ltd, article No.: MD106-01).
Embodiment 2
The small extraction reagent kit of plasmid (TIANprep Mini Plasmid Kit) produced using TIANGEN company, article No.:
DP103, concrete composition: RNase A, Buffer P1, Buffer P2, Buffer P3, Buffer PD, Buffer PW,
Buffer EB、Spin Columns CP3、2 ml Collection Tube。
(1) S. aureus Inoculate of purpose plasmid (plasmid contains anti-kanamycin gene) will be contained in 5ml
In BHI culture medium containing 15 μ g/ml kanamycins (KAN), 37 DEG C of shaking table culture 11 hours a small amount of amplification bacterium solutions；
(2) it is added in 250ml culture bottle in BHI broth of the 100ml containing 15 μ g/ml KAN, is inoculated with the first of 1ml step (1)
Grade bacterium solution is into culture bottle, 37 DEG C of shaking table cultures, 14 hours amplification bacterium solutions；
(3) the staphylococcus aureus bacterium solution for collecting 100ml step (2) carries out room temperature centrifuge separation, and the revolving speed of centrifuge separation is
6000 revs/min, the time of centrifuge separation is 13 minutes, abandons culture supernatants, and centrifuge tube left-hand thread is residual in blotting paper exhaustion
Liquid；
(4) take the reagent Buffer P1/RNase A of 3ml cracking microorganism in the 5ml containing lysozyme and staphylococcus enzyme
In centrifuge tube, make the concentration 5mg/ml of lysozyme, the concentration of staphylococcus enzyme is 1mg/ml(lysozyme and staphylococcus lysozyme
In Keyinbio company, article No. is respectively 12650-88-3 and 9011-93-2 for purchase)；
(5) mixed liquor of 4ml step (4) the cracking staphylococcus aureus is taken to be added to step (3) described equipped with golden yellow
In the centrifuge tube of staphylococcus precipitating, being vortexed to be resuspended to handle to thallus is all resuspended, and without precipitating in centrifuge tube, 37 DEG C of warm bath 25 are divided
Clock；
(6) 510 microlitres of step (5) re-suspension liquids are shifted respectively into new 2mL centrifuge tube, are separately added into 200mg glass powder (0.1-
0.6mm, high-temperature sterilization), it 2700 revs/min, is vortexed and mixes 9 minutes, vortex mixer input power is 1.2W, output power
For 0.8W, it is completely severed the cell wall of staphylococcus aureus；
(7) 510 microlitres of alkaline lysis liquid Buffer P2 are added in each centrifuge tube in step (6), overturn centrifuge tube 5-10
It is secondary, make obtained cellular lysate in step (6)；
(8) 11 microlitres of 22mg/ml Proteinase Ks (purchase is in Merck KGaA company, article No.: 39450-01-6) to step (7) is taken
It in resulting lysate, is mixed by inversion centrifuge tube 7 times, is stored at room temperature 10 minutes, is mixed by inversion 6 times and obtains at intervals of two minutes therebetween
Mixed liquor containing Proteinase K；
(9) it takes 420 microlitres of rapid precipitation buffer solution B uffer P3 into step (8) resulting lysate, is mixed by inversion 13 times and allows
Solution thoroughly neutralizes；
(10) suspension obtained by step (9) is subjected to room temperature centrifuge separation, the revolving speed of centrifuge separation is 12500 revs/min, centrifugation
The isolated time is 12 minutes；
(11) 6 adsorption columns (adsorption column used in embodiment 2 is Spin Columns CP3) are placed in 6 collecting pipes (2
Ml Collection Tube) in, transfer step (10) resulting supernatant is in adsorption column respectively, room temperature centrifuge separation, from
The revolving speed of heart separation is 9000 revs/min, and the time of centrifuge separation is 45 seconds, abandons filtrate, adsorption column is recovered in collecting pipe；
(12) it takes 620 microlitres of rinsing liquids, 1 Buffer PD in the adsorption column of step (11), stands 4 minutes, room temperature centrifugation point
From the revolving speed of centrifuge separation is 9000 revs/min, and the time of centrifuge separation is 45 seconds, abandons filtrate, adsorption column is recovered collection
Guan Zhong；
(13) take 640 microlitres of rinsing liquids, 2 Buffer PW in the adsorption column of step (12), room temperature centrifuge separation, centrifuge separation
Revolving speed be 9000 revs/min, time of centrifuge separation is 40 seconds, abandons filtrate, adsorption column is recovered in collecting pipe；
(14) step (13) are repeated；
(15) room temperature step with centrifugal separation (14) resulting adsorption column, the revolving speed of centrifuge separation are 12500 revs/min, centrifugation point
From time be 4 minutes, dry adsorption column removes ethyl alcohol；
(16) an absorption column sleeve in step (15) is taken 80 microlitres of elution buffers in the centrifuge tube of the 1.5ml of sterilizing
Buffer EB to adsorption column film center, stand 1 minute, room temperature be centrifuged eluted dna, be centrifuged elution revolving speed be 12500 turns/
Minute, the time of centrifuge separation is 2 minutes；
(17) product that step (16) elute is transferred in another adsorption column of step (15) and is eluted, operating procedure with
Step (16) is identical；
(18) and so on until the elution of all adsorption columns completely, obtains high-purity plasmid DNA；
(19) adsorption column in (18) is discarded, (18) resulting eluent is stored in -20 DEG C.
(20) it detects: using mass percent concentration to carry out for 0.8% Ago-Gel the Plasmid DNA product of elution
Detection, buffer are that (TAE buffer is made of TAE buffer trishydroxymethylaminomethane, acetic acid and ethylenediamine tetra-acetic acid
Buffer), voltage be electrophoresis 90min under conditions of 1000V, as a result can refer to Fig. 1, extract aureus plasmid
DNA as a result, electrophoretic band from left to right successively are as follows: Plasmid DNA (English name of plasmid be plasmid), marker
Marker(is bought from Bo Maide Bioisystech Co., Ltd, article No.: MD106-01).
Embodiment 3
The small extraction reagent kit of plasmid (QIAGEN Plasmid Mini Kit) produced using QIAGEN company, article No.: 12143, tool
Body composition: RNase A, Buffer P1, Buffer P2, Buffer P3, Buffer QC, Buffer QF, QIAGEN-tip, 2
ml Collection Tube。
(1) will contain the S. aureus Inoculate of purpose plasmid (the purpose plasmid contain anti-kanamycin gene) in
5ml contains in the BHI culture medium of 15 μ g/ml kanamycins (KAN), 37 DEG C of shaking table culture 12 hours a small amount of amplification bacterium solutions；
(2) it is added in 250ml culture bottle in BHI broth of the 150ml containing 15 μ g/ml KAN, is inoculated with the first of 3ml step (1)
Grade bacterium solution is into culture bottle, 37 DEG C of shaking table cultures, 16 hours amplification bacterium solutions；
(3) the staphylococcus aureus bacterium solution for collecting 100ml step (2) carries out room temperature centrifuge separation, and the revolving speed of centrifuge separation is
8000 revs/min, the time of centrifuge separation is 15 minutes, abandons culture supernatants, and centrifuge tube left-hand thread is residual in blotting paper exhaustion
Liquid；
(4) take the reagent Buffer P1/RNase A of 5ml cracking microorganism in the 10ml containing lysozyme and staphylococcus enzyme
In centrifuge tube, make the concentration 5mg/ml of lysozyme, the concentration of staphylococcus enzyme is 1mg/ml.Lysozyme and staphylococcus lysozyme
In Keyinbio company, article No. is respectively 12650-88-3 and 9011-93-2 for purchase；
(5) mixed liquor of 5ml step (4) the cracking staphylococcus aureus is taken to be added to step (3) described equipped with golden yellow
In the centrifuge tube of staphylococcus precipitating, being vortexed to be resuspended to handle to thallus is all resuspended, and without precipitating in centrifuge tube, 37 DEG C of warm bath 30 are divided
Clock；
(6) 500 microlitres of step (5) re-suspension liquids are shifted respectively into new 2mL centrifuge tube, are separately added into 200mg glass powder (0.1-
0.6mm, high-temperature sterilization), it 2800 revs/min, is vortexed and mixes 10 minutes, vortex mixer input power is 1.2W, output work
Rate is 0.8W, is completely severed the cell wall of staphylococcus aureus；
(7) 500 microlitres of alkaline lysis liquid Buffer P2 are added in each centrifuge tube in step (6), overturn centrifuge tube 10 times,
Make obtained cellular lysate in step (6)；
(8) 12 microlitres of 20mg/ml Proteinase Ks (purchase is in Merck KGaA company, article No.: 39450-01-6) to step (7) is taken
It in resulting lysate, is mixed by inversion centrifuge tube 10 times, is stored at room temperature 10 minutes, is mixed by inversion 6 times and obtains at intervals of two minutes therebetween
Mixed liquor containing Proteinase K；
(9) it takes 450 microlitres of rapid precipitation buffer solution B uffer P3 into step (8) resulting lysate, is mixed by inversion 15 times and allows
Solution thoroughly neutralizes；
(10) suspension obtained by step (9) is subjected to room temperature centrifuge separation, the revolving speed of centrifuge separation is 13000 revs/min, centrifugation
The isolated time is 10-15 minutes；
(11) 6 adsorption columns (adsorption column used in embodiment 3 is QIAGEN-tip) are placed in 6 collecting pipe (2 ml
Collection Tube) in, transfer step (10) resulting supernatant is in adsorption column respectively, room temperature centrifuge separation, centrifugation point
From revolving speed be 10000 revs/min, time of centrifuge separation is 30-60 seconds, abandons filtrate, adsorption column is recovered in collecting pipe；
(12) it takes 630 microlitres of rinsing liquids, 1 Buffer QC in the adsorption column of step (11), stands 5 minutes, room temperature centrifugation point
From the revolving speed of centrifuge separation is 10000 revs/min, and the time of centrifuge separation is 30-60 seconds, abandons filtrate, adsorption column is recovered
In collecting pipe；
(13) take 650 microlitres of rinsing liquids, 2 Buffer QF in the adsorption column of step (12), room temperature centrifuge separation, centrifuge separation
Revolving speed be 10000 revs/min, time of centrifuge separation is 60 seconds, abandons filtrate, adsorption column is recovered in collecting pipe；
(14) step (13) are repeated；
(15) room temperature step with centrifugal separation (14) resulting adsorption column, the revolving speed of centrifuge separation are 13000 revs/min, centrifugation point
From time be 5 minutes, dry adsorption column removes ethyl alcohol；
(16) an absorption column sleeve in step (15) in the centrifuge tube of the 1.5ml of sterilizing, take 100 microlitres of aqua sterilisas to suction
The film center of attached column, stands 3 minutes, and room temperature is centrifuged eluted dna, and the revolving speed for being centrifuged elution is 13000 revs/min, centrifuge separation
Time be 2 minutes；
(17) product that step (16) elute is transferred in another adsorption column of step (15) and is eluted, operating procedure with
Step (16) is identical；
(18) and so on until the elution of all adsorption columns completely, obtains high-purity plasmid DNA；
(19) adsorption column in (18) is discarded, (18) resulting eluent is stored in -20 DEG C.
(20) it detects: using mass percent concentration to carry out for 0.8% Ago-Gel the Plasmid DNA product of elution
Detection, buffer are that (TAE buffer is made of TAE buffer trishydroxymethylaminomethane, acetic acid and ethylenediamine tetra-acetic acid
Buffer), voltage be 1000V under conditions of electrophoresis 90min, as a result can refer to shown in Fig. 1, extract staphylococcus aureus matter
Grain DNA as a result, electrophoretic band from left to right successively are as follows: Plasmid DNA (English name of plasmid be plasmid), marker
Marker(is bought from Bo Maide Bioisystech Co., Ltd, article No.: MD106-01).
In addition, three embodiments of the invention are also had detected with spectrophotometer and are extracted other than electrophoresis runs glue detection
The purity of the Plasmid DNA arrived.Wherein, the OD260/OD280 value for the Plasmid DNA that embodiment 1 is extracted is 1.88, and embodiment 2 is extracted
Plasmid DNA OD260/OD280 value be 1.83, embodiment 3 extract Plasmid DNA OD260/OD280 value be 1.85.Three
Embodiment extracts to obtain the OD260/OD280 of Plasmid DNA near 1.8, indicates very high (its sequence such as sequence table of DNA purity
1).
Three case study on implementation can successfully extract aureus plasmid, and the time used is close, pass through electrophoresis knot
Fruit (Fig. 1) it is found that this method can successfully extract target plasmid and be applicable to various routine operations, including digestion,
PCR, sequencing, connection, conversion, library screening, In Vitro Translation, some conventional passage cells of transfection etc..
Compared with prior art, method of the extracting method provided by the invention due to combining physical wall breaking, molten grape ball
The dosage of bacterium enzyme reduces one third, and chemical broken time shortens one hour, can reach preferable cracking in a short time
The effect of cell wall promptly releases Plasmid DNA from staphylococcus aureus, there is good extraction effect.
Above embodiments are only preferrred embodiment of the present invention, for explaining only the invention, are not intended to limit the present invention, this
Field technical staff should belong to guarantor of the invention without departing from change made under spirit of the invention, replacement, modification etc.
Protect range.
Sequence table
<110>South China Science & Engineering University
<120>a kind of method for extracting aureus plasmid
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 7777
<212> DNA
<213>haemophilus parasuis (Glaesserella parasuis)
<400> 1
actagcaggc tgcggattag acgttaccga atgaacagaa ttgggcgaac aagccgttaa 60
tgtcagcaat aataatgtgc cgatttttaa cttatttaac ataaattatc ctttgatttt 120
actaaataaa tttcttttca ggcagcctga aacgtacatt gcgcggaaat tcggataatt 180
tgtttgtaga acaaaaaaac agattttggc gaatttaagc gcaaaagtct agcaatttag 240
ccttataaaa cagaaaaagg caatgatatt tctatcactg cctttttcct ttagcggtct 300
atgccgataa ataaaataag cggtctaagc cgaacaactt cattatatgg ctttatggtt 360
cattattcaa gctatcaagt attcttttac cactcacggc aataagcact tttttctgaa 420
tgtttgtaac tacgtcatca ggcaattttt gatttagttt tttgccatta tcaaaaatgt 480
aatacagacg ttttttactt acacagcgga cgtgttcagt cttaacccat cgttctcttt 540
tctcatagaa gctagtttct tggatcagtt caggctttaa atactcgtga aaagtggcta 600
aacgtccgta ggcttttgta gatgaaatag gggcaacaac cacgctataa ccgtcaatat 660
cgccatttag cacaaccaca aggcggcgtt tacgcatttc aggtggaata tgtccgttta 720
ctgtttcagg ttcagaccat tgcccgaaat cacattccaa cagttcgcca actttggggc 780
ggtggttaat tgccattttt actgctcctt agtaccacgt ttgtacatca ttctaacgtt 840
atcttccgtc atattcagca tttcggcaat tctagcccac ttcacaggcg gataagtatt 900
tcgtaatgtt tgaatctcgt gcaataaacg agctttttca tctttcaaag tgccgtgttc 960
ttttcggatt tggcttgcat aggttgaaat agttttttca gctacttcaa atttttcagt 1020
taaaaggatt ctttctgcct tgctgtattt atgattcggt ccgtatgtgt ctaacatata 1080
cgtttttatt tggttcattt tgtctagtct ccgctgttgg taacttggat atttacagac 1140
taattcattt ctgaaacagt atttgacaat gcttctaatc attgctttta cttcgttttc 1200
aggtagtcca atctctctat ttttactgtt cgctacattg aacaggtatg taaaaaattc 1260
ttttgtcgct ttggtttcca catattcaga gcttacttca taggcaagta accgcatttc 1320
atcaaataat tggcaattac gagaattttc atcgtattta taatgctcat ttggttctac 1380
ttcggtcaaa atgtgatttt ctatatcagt aaaatctaca tcagcaaatt taggattgta 1440
tctatccgca aaagctcgat taaataattc atcaaccgag taagggggaa gttcagtcca 1500
cgcaacagcc ccaacttccg tagcaaaaaa aggatttttg gcattatgta acttgaaatt 1560
tgtatcgcca ttaaataatt cattcagtct tatcactgtt tcaatgaatt ttatctgttt 1620
agattggtta tctagtggta acggctcgcc atttttatcc aattttctat aatcaaaaaa 1680
atctttatct tctagcttaa atactggtgt agcaaggaca tagccaactt gtagggactt 1740
ttgtttttta ggatttctaa tcaaataatt aggcgttagg tcaaacttct caaaaacgct 1800
gtaatcaccg tcaaaatcat caatatcaac gattaaaaca gggaataatt ccgtagcgtt 1860
gttgtatcta tcggtaaatt taacggtaaa agtgaagtgt gtatattctg aaaacgccct 1920
tccccagttc ttactatttg aatagatttt tacaaattta ctaaatgagc cgtctttttg 1980
ctttcctgta ctggcacgca aaacagttct attcataaaa ttagaaaata attttgcaca 2040
agtttcatta ttaaaaatat gagtattatt ttgaataatt ggactataca aacttaatgg 2100
gtttctagcg ttgtggaagc gttgttcttc cattgtgatg aagtcaaaat actttacttt 2160
aaagtaaagt tcgggtatta tttgtttcat aaattcagtg aacccttaga ttgtgtctag 2220
tcgccaaact taacagtcta ggggttttct ttttggttaa tttttagttg tcgggcagtg 2280
tatcacattt agaaaaagcg aacaatatcc ataaatttaa cactgtttat tttttgaaca 2340
aaaccgaaca aaccaccctt actatcatat atatactttt ggctctcttc tgccgtccgt 2400
tttttgtctc tgttttcgtg tccttctctc tgtatttccg aacattcttt ccttgcttct 2460
tagcgtgatg tagttccgtt cgttcagttc gtaaactcgg tcgtaaatcc actgctgaac 2520
gcagtgtctt tgctcccttg tttgctcact tctctcactc ttgtttttgg ttgttttttg 2580
atctacactt gtattcagta ttttaagcga gcatagattt gtacgttact tcgtaacaac 2640
aaatcgctcg taaagggtgg tgctacgccc ccctttaatc ccccagtttt agatcggagc 2700
atttaatcaa tggcaaagaa aggtattaaa cgtggtgtat ctagtcgttt agagattcga 2760
ttaccagtag aaacaaaaca gaaattactt gatatgtgtg gggatcactt ttctacgtct 2820
gaaatgttaa ggcagattat tgagcgtggt gaagttcctg atttaaccgc tacatcttgt 2880
attagagaga aaaaagccac tcttgagcct ttgattattg agcttgctag ggttggcaat 2940
aacttaaatc agataacaaa ggcttctaat gctacattgc ggtttttcaa tcattacaat 3000
acgcaagaca ttgataaatc agcattaggg ttagttgcat tacagcggtt aaatgatgaa 3060
atggctagtt tatttgaaac tagaaaatca ttgaatcgtt tgatttctga tttagttaaa 3120
tggagtaata gcaatgctag ttaaattttt taatactgga aagggatcag ctaggcacgc 3180
atttgattac ttactgaata atgaatgtgt taaaaacggt acagcaaagc tagttaaagg 3240
taatcctgat gtaattacgt ttctaacaga gcaacgaatg aaagagccta gttatagggg 3300
gggagctttg tacacatcag gggtactgtc gttttcgcct gaagaatcta agtctttgac 3360
tgattctaat ctcggtgcaa tcattgggcg atttgaggct actatattcc ctaaattaga 3420
tataactagg tttaatattg cttgggttct tcataaagat aaggatcgta cagaattgca 3480
ttttgtgatt cagaattttg atttagagac taaaaaagca tttacgcctt ttgttgccga 3540
ccgtgattta tcaagggtaa atcaattcaa agataaggtt aatgctgatt ttaatttaag 3600
tgatcctaat ttggtaaaat cacattacca cgttaaaggt ttgaaacggt tatcggaaga 3660
acaaaaatca ttttatcgtg aattgaataa agcctatgcc caatatgttg aagaaagaga 3720
aaatccatct ttcataaaac aagctaaggg cattttatcc aaaataatgg ggcaaaatga 3780
gccacaggca cacgatttac attcaaatga agataagtac gcctttttgc aaaaattcat 3840
tacagagcgt tttaaggggc ttaaaatcaa tcgctcatct gataaatatg catcggttga 3900
ttttaacggt tcaaaaatgc gtttattcta tgaaaaattc gtggttaaag agcttgatga 3960
cgcttttaaa attaaagttg agcagaacaa agaaaaagga ttattgccgg tagatagcgg 4020
acctattttt gcagagttac ttcaatcata tcgccaagca attcaacagg caaaagctga 4080
aaaagaacgc ttaaaacgtg cggaagaaga agcagaaaaa gagcgtttac gccttgtagc 4140
acttgagaaa cagaaagaat tagagaataa atttagagca attttgaatg gttcaaaaga 4200
tgtatttatt gaaaaagtta tcaaaaaggt tattgatcag gactataaga cacgttgcaa 4260
tgagttatta gagcctacat ggatggggac taataaaatt cacaatttta tttgttactc 4320
taaagagctt tcttctgatg atttgaacgc tgaaatgaat aaaattaaac aggcagtaga 4380
acaagaattt ggaaaaacac gtttagatga gcttgaagtt aaaggatttt tcaatcttca 4440
atctgctatt tctaaattag aaactatgca agaaactaca ttagtcaaat tgaaagatgt 4500
gtacattgat ttttataaaa aggtagatac agctagattt aataagttta aagcagaagt 4560
tgatgaatat aaatcaactc atagaagtaa cgctgttatg cttgaatata gagattatgc 4620
aacagccttt ttaaatgatt tattaccaag aatggaacgt tctctaactg tttcaaatcc 4680
tagcgtggaa gcagaaaaga caagtgttta tcaaccacag ccacaagcga agccgaacgc 4740
accaaaattc agaatgtagg gggactgaat acggagaacg ggaatcaaat ccccccacgt 4800
tttagccgca gaaaaatcct gatatttgct cttgaattgt caaaccaagt ccttgaattg 4860
tcaaaccaag tgcaacgatt ttttgaagta aacttcataa ggtgttttcc aacctaaaca 4920
tttacgtggt cgtaaattca gtttattgat cacctgttga atatcaactt cgctccactg 4980
attgatgtct tggtgcttcg gaaagtattc acgaagtaac ccatttgtat tttcattcgt 5040
tcagctcgcg agaaatggtg ctggagctac gtcccagtgc tttggcaatt tctgcttgtt 5100
ttttgccctg tgcgagtaaa atcattatct tttctcgctc gtttattgta agatgtcggt 5160
aggaagtact catcatttgg aagtggtttt tgtgtggaaa caaaattata ttctagttga 5220
tgagtacttc tttttttgtt gcacttggat tgtaaattca gccatttttt taaactattt 5280
aatactaatg tcttttataa tagcttttca tatattatat aatcaatctt tataagtcct 5340
tttataaatt tcttttctac cattttcgat aaattcctgt ttaatatttt taattccata 5400
aacaatagtt tcaataggat aatattcttc aactatatct tgatattctt ttgctttctc 5460
aatatctata tttccataca ttcttaatat atcttctcca aaatttgttc ctatttcttc 5520
ttcactatct tcaagtaagt atataaaatc acaatattca tctataattc cagaatctcc 5580
aaaatcaatt attccagtta atctattatt gccatctaac aatagatgat tacaactaaa 5640
atcattatgg cataaacact ttttaccctc aaaaactgtt gttgcattta gtctttccat 5700
aaaactttct atataatctt tttctatatc agttaaatca ttataaatag tttcacgcaa 5760
caatatatac tcttctaata cattttgttt attatcaata gtacattcac taatatctgt 5820
ataatctaaa ccgtgcattt gtcttaaaaa actggcaata tctcgtttta acaaattttg 5880
ttcttcttct gacatagtag aataaatttc tggtgttaaa aaagttcctt taatttcttt 5940
ataacctagt atagataatt catcactaat atacgaatat tcaatattag gaatttttac 6000
attagtttct aaatttgtat ttaaaaaatt atatattgct ttttcttttg cataaccttt 6060
tttcttatta gtactaaatt ttgttttaaa aatgtattca ttattaacta aatatgccac 6120
actatcataa ccactaccga ttatttcaat actatctact ttgaaattat caaagtaatg 6180
ctcaattaaa tatttcattg ccttaacatt tgtggcatta tcatcatatc tatattccat 6240
taaataacaa tcttcttttt tgccctcgtg taattcatgt tctggcaaat cttcaataat 6300
tctaaaacca gatttttggt atgcccttat tgctcttgga ttatttttat gagggtctaa 6360
aataactgca ttagcatttc tttctttttt caaaaattca aaaatcaatt taatatatct 6420
tgtaccaatt cctttactcc aataatttgg ctctcctata aattgatcca taccatagac 6480
tatctcatca gtttttggat aatgataatc agtatataac tcatcataca ttttatatat 6540
ttgtccatat ccaataggaa cattgttata ttcaataatt actctaaaaa cttcatcttc 6600
ccaaggctct gtataatgtt tttttaatga ttctaatgta tattttttat ctctaccacc 6660
ataaaattct aatactcttt catcagttaa ccattttaac atcaaaggaa aatcatcatc 6720
tattaaagtt cttatacata tttcattttc aactatattc atttatttat cacctttttc 6780
ataatcatat acatatacta tttcatcttt ataatcattt ttaccaccta atttttcata 6840
tacatggcaa gctctaggat tacctttatc agttattaaa aacatttcag aacaaccaat 6900
ctctttagaa tattccttaa taaaaggctg aattttcact ttttctaaca aaatgaattt 6960
gttagttgag ctgtaaagta tgaaatactt gctttgctgc atcttctact aatttattgt 7020
tgaatttggc ttcttcggta aattgcgtac tcatgattgc catcacaatc ggtttgcgat 7080
ttggtatgcg aaccaccgca atatcattgc gtacaccata tttacccgcc ccgcttttat 7140
cgtacacttt ccacgatgtt ggcgtagcag cgcgaatcaa tggattgcct gttgcgttat 7200
tgtccaacca attccacaaa atcgtttttt gcgattcggt taatgtgttg cccaataaat 7260
acgcatttaa attcatcgcc atttgtttgg gtgtactcgt atcacgaata tcgttgggtt 7320
tggcttgatt taaatcgggt tctagccgat tggtatgggt tacgttatcg cctaattgtc 7380
gcaaaatacg ttgatattgt tccacgccac ccaattcttt gagcagcaaa ttggtcgcgc 7440
tgttgtcgct aaaccgcacg gctgcttcac ataattgggc aatcgtcatg cctttgccaa 7500
cgtatttttg ggtttcggga gaataactaa ccaaatcttt ttggctatat gaaatggtac 7560
gatttaaatc tttttcaggc agcgattgca acaccgcccc agccaacaac gccttgaaag 7620
tggacgcata agcaaagcgt tcatctgcac gataagacaa agaatgtccc gtttctgtat 7680
cccatacata aacgccaatt cgggcttgat actgctgttc caaattcgcc aaagtctgtt 7740
gaaaggtggc ttgtgtggct gattgttgca caggcgc 7777
Claims (10)
1. a kind of method for extracting aureus plasmid, which comprises the steps of:
(1) by the S. aureus Inoculate of the plasmid containing purpose in brain heart oxoid/Antibiotic medium, shaking table culture,
Bacterium solution is expanded, primary bacterium solution is obtained；
(2) primary bacterium solution described in inoculation step (1) is into brain heart oxoid/Antibiotic medium, shaking table culture, expands bacterium
Liquid obtains secondary bacterium solution；
(3) secondary bacterium solution described in step (2) is subjected to room temperature centrifuge separation；It falls to abandon culture supernatants after centrifugation, centrifuge tube
Left-hand thread obtains the centrifuge tube equipped with staphylococcus aureus precipitating in exhausting raffinate on blotting paper；
(4) lysozyme and staphylococcus lysozyme are added in the reagent of cracking microorganism；Obtain cracking Staphylococcus aureus
The mixed liquor of bacterium；
(5) mixed liquor of step (4) the cracking staphylococcus aureus is added to described in step (3) equipped with golden yellow grape
In the centrifuge tube of coccus precipitating, be vortexed be resuspended processing to thallus be all resuspended, in centrifuge tube without precipitating until, then at constant temperature bath
Reason, obtains re-suspension liquid；
(6) re-suspension liquid described in step (5) is transferred in another centrifuge tube, glass powder is added, vortex is uniformly mixed so as to obtain containing glass
The re-suspension liquid of glass powder；
(7) alkaline lysis liquid is added in the re-suspension liquid containing glass powder described in step (6), covers centrifuge tube lid, overturn centrifuge tube
5-10 times, obtain mixed liquor；
(8) Proteinase K is added in step (7) described mixed liquor, covers centrifuge tube lid, reverse centrifuge tube 5-10 times, so
After be stored at room temperature 10-15 minutes, be mixed by inversion 5-10 times every 2-3 minutes therebetween and obtain the mixed liquor containing Proteinase K；
(9) rapid precipitation buffer is added in the mixed liquor containing Proteinase K described in step (8), covers the lid of centrifuge tube,
It is 10-15 times reverse, obtain suspension；
(10) suspension for obtaining step (9) carries out room temperature centrifuge separation, obtains supernatant；
(11) adsorption column is placed in collecting pipe respectively, supernatant obtained by step (10) is added in adsorption column respectively, room temperature from
Heart separation abandons the filtrate in collecting pipe, adsorption column is recovered in collecting pipe；
(12) by rinsing liquid 1 in adsorption column, room temperature is stood, and room temperature centrifuge separation abandons filtrate, adsorption column is recovered collecting pipe
In；
(13) by rinsing liquid 2 in adsorption column, room temperature centrifuge separation abandons filtrate, adsorption column is recovered in collecting pipe；
(14) step (13) are repeated；
(15) room temperature centrifugation step (14) resulting adsorption column；
(16) by the absorption column sleeve after centrifugation in sterilized centrifuge tube, adsorption column is added in elution buffer or aqua sterilisa
Film center, room temperature centrifugal treating after standing discards adsorption column, an eluent is obtained in centrifuge tube, in an eluent
Contain the purpose plasmid；
(17) step (16) described eluent is transferred in another adsorption column of step (15) and is eluted, room temperature after standing
Centrifugal treating discards adsorption column, and secondary eluent, the purpose matter increased in secondary eluent containing concentration are obtained in centrifuge tube
Grain；
(18) and so on until the elution of all adsorption columns completely, obtains final eluent, that is, is enriched with the elution of purpose Plasmid DNA
Liquid；
(19) eluent that purpose Plasmid DNA is enriched with described in step (18) is placed on stored refrigerated in refrigerator.
2. the method according to claim 1, wherein purpose plasmid described in step (1) contains anti-antibiotic
Gene；The temperature of shaking table culture described in step (1) is 37 DEG C, and the time of shaking table culture is 10-12 hours；Institute in step (2)
The dosage for stating primary bacterium solution is 1-3mL, i.e., inoculum concentration is 1-3mL；Brain heart oxoid/Antibiotic medium described in step (2)
Dosage be 100-150mL；The temperature of shaking table culture described in step (2) is 37 DEG C, and incubation time is 12-16 hours.
3. the method according to claim 1, wherein the volume of secondary bacterium solution described in step (3) is 60mL-
100mL；The rate of the centrifuge separation of room temperature described in step (3) is 6000-8000 revs/min, and the time of centrifuge separation is 10-15
Minute.
4. the method according to claim 1, wherein the additional amount of lysozyme described in step (4) is every milliliter
It cracks in the reagent of microorganism and adds 5-8mg；The additional amount of staphylococcus lysozyme described in step (4) is that every milliliter of cracking is thin
1-3mg is added in the reagent of bacterium thallus；The additional amount that the mixed liquor of staphylococcus aureus is cracked described in step (5) is 3-
5mL；The temperature of the processing of constant temperature bath described in step (5) is 37 degrees Celsius；The time of constant temperature bath processing is 20-30 minutes.
5. the method according to claim 1, wherein the dosage of re-suspension liquid described in step (6) is that 500-520 is micro-
It rises；The partial size of glass powder described in step (6) is 0.1-0.6mm, and the glass powder is using preceding needing high-temperature sterilization, the glass powder
Additional amount be 180-200mg；Be vortexed described in step (6) mixing vortex time be 8-10 minutes, the vortex for the mixing that is vortexed
Rate is 2600-2800 revs/min；The additional amount of alkaline lysis liquid described in step (7) is 500-520 microlitres.
6. the method according to claim 1, wherein Proteinase K described in step (8) is a kind of from Candida albicans
The strength protein resolvase that bacterium is separated；The concentration of Proteinase K is 20-25mg/mL, and the additional amount of Proteinase K is that 10-12 is micro-
It rises.
7. the method according to claim 1, wherein the additional amount of rapid precipitation buffer described in step (9)
It is 420-450 microlitres；The rate of the centrifuge separation of room temperature described in step (10) is 12000-13000 revs/min, room temperature centrifugation point
From time be 10-15 minutes；The rate of the centrifuge separation of room temperature described in step (11) is 8000-10000 revs/min, room temperature
The time of centrifuge separation is 30-60 seconds.
8. the method according to claim 1, wherein the additional amount of rinsing liquid 1 described in step (12) is 600-
630 microlitres；The time that room temperature described in step (12) is stood is 3-5 minutes；The speed of the centrifuge separation of room temperature described in step (12)
Rate is 8000-10000 revs/min, and the time of room temperature centrifuge separation is 30-60 seconds；The addition of rinsing liquid 2 described in step (13)
Amount is 620-650 microlitres；The rate of step (13) the room temperature centrifuge separation is 8000-10000 revs/min, room temperature centrifugation point
From time be 30-60 seconds.
9. the method according to claim 1, wherein the centrifugal rotational speed of the centrifugation of room temperature described in step (15) is
12000-13000 revs/min, the time of centrifugation is 3-5 minutes；Elution buffer described in step (16) or aqua sterilisa plus
Entering amount is 60-100 microlitres；The time of step (16) and step (17) described standing is 1-3 minutes；Step (16) and step (17)
The rate of the room temperature centrifugal treating is 12000-13000 revs/min, and the time of centrifugation is 1-2 minutes.
10. the method according to claim 1, wherein cracking the reagent of microorganism, step described in step (4)
Suddenly alkaline lysis liquid described in (7), rapid precipitation buffer described in step (9), adsorption column described in step (11), step (12)
Described in rinsing liquid 1, elution buffer described in rinsing liquid 2 described in step (13) and step (16) derive from plasmid and mention
Take kit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN113322304A (en) * | 2021-06-19 | 2021-08-31 | 江苏先声医学诊断有限公司 | Clinical sample processing method applied to second-generation infection metagenome detection |
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| CN103205417A (en) * | 2013-04-23 | 2013-07-17 | 天根生化科技（北京）有限公司 | Method for quickly extracting high-purity plasmid DNA (Deoxyribonucleic Acid) |
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| CN103205417A (en) * | 2013-04-23 | 2013-07-17 | 天根生化科技（北京）有限公司 | Method for quickly extracting high-purity plasmid DNA (Deoxyribonucleic Acid) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113322304A (en) * | 2021-06-19 | 2021-08-31 | 江苏先声医学诊断有限公司 | Clinical sample processing method applied to second-generation infection metagenome detection |
| CN113322304B (en) * | 2021-06-19 | 2022-06-07 | 江苏先声医学诊断有限公司 | Clinical sample processing method applied to second-generation infection metagenome detection |
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Application publication date: 20190419 |