CN108866043A - A kind of pretreatment reagent kit and method of bacterial plasmid extracting - Google Patents
A kind of pretreatment reagent kit and method of bacterial plasmid extracting Download PDFInfo
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Abstract
The present invention relates to a kind of pretreatment reagent kits of bacterial plasmid extracting of the present invention, it includes following component：Component A：100~200mg/ml lysozyme；B component：Cleaning solution；The component A is 100mg/ml lysozyme.The invention also discloses the pre-treating methods of bacterial plasmid extracting.The present invention also provides a kind of bacterial plasmid extraction agent box and methods.The present invention provides the pretreatment reagent kits and method of a kind of extracting of bacterial plasmid, the kit and method can effectively improve the extraction efficiency of bacterial plasmid, it is especially very good to the extraction effect of lactobacillus plantarum, it can overcome the problems, such as that existing method can not effectively extract Gram-positive bacteria plasmid, have a vast market application prospect.
Description
Technical field
The present invention relates to the pretreatment reagent kits and method of a kind of extracting of bacterial plasmid.
Background technique
Plasmid be can in living cells self-replacation nucleic acid molecules.Plasmid-mediated Horizontal Gene Transfer is considered as
The main drive of Phylogenetic diversity of bacteria and adaptation.With the continuous deepening of research, plasmid has been widely used in gene studies work
In tool.The extraction of Plasmid DNA is a key technology in molecular biology, is the molecules skills such as clone, transfection and gene therapy
Essential link in art.Research finds that new plasmid has very important effect from bacterium.
For gram-positive bacteria due to containing peptide glycan in cell wall, the degree of cross linking is high, it is difficult to crack, lead to gram-positive bacteria
Plasmid extract extremely difficult, one or several a natural plasmids in especially many lactobacillus plantarum strains, extraction is even more
It is difficult.Bead is reported in existing literature to be facilitated to overcome cracking difficult in conjunction with short vortex time, but complicated for operation, cost
It is high.
Summary of the invention
In order to solve the above-mentioned technical problems, the present invention provides a kind of pretreatment reagent kit of bacterial plasmid extracting and sides
Method.
The pretreatment reagent kit of bacterial plasmid extracting of the present invention, it includes following component：
Component A：80-160mg/ml lysozyme；
B component：Cleaning solution；
The component A is 100mg/ml lysozyme.
The cleaning solution is 4~6% glucose solutions or solution I；The solution I be added with 10mmol/L EDTA,
The Tris-HCl solution of 50mmol/L glucose, 100 μ g/ml RNase A, wherein the concentration of Tris-HCl solution is 25
mmol/L。
The concentration of the glucose solution is 5%.
The present invention also provides a kind of bacterial plasmid extraction agent boxes, it wraps following component：
(1) pretreatment reagent kit above-mentioned；
(2) plasmid extraction kit.
The component (two) is the kit of alkaline lysis method of extracting plasmid.
The kit of the alkaline lysis method of extracting plasmid includes following ingredient：
Solution I：Added with 10mmol/L EDTA, 50mmol/L glucose, 100 μ g/ml RNase A Tris-HCl
Solution, wherein the concentration of Tris-HCl solution is 25mmol/L；
Solution II：Concentration added with 1% (W/V) SDS is the NaOH solution of 250mmol/L；
Solution III：The mixed solution of potassium acetate and acetic acid, the concentration 3mol/L of potassium acetate, the concentration of acetic acid are 5mol/L；
Solution IV：Every 1L contains 10mmol Tris-HCl and 0.80L ethyl alcohol, remaining is water (pH7.5)；
Solution V：The Tris-HCl solution of 10mmol/L.
The present invention also provides a kind of pre-treating methods of bacterial plasmid extracting, it is any one using Claims 1 to 4
Pretreatment reagent kit processing described in, steps are as follows：Microorganism is handled with component A, is then washed with B component.
The present invention also provides a kind of bacterial plasmid method for extracting, it is extracted using kit above-mentioned, and steps are as follows：
Pre-treatment is carried out according to preceding method, reuses plasmid extraction kit extraction.
The present invention provides a kind of pretreatment reagent kits of bacterial plasmid extracting and method, the kit and method to have
Effect improves the extraction efficiency of bacterial plasmid, especially very good to the extraction effect of lactobacillus plantarum, can overcome existing side
Method can not effectively extract the problem of Gram-positive bacteria plasmid, have a vast market application prospect.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes further specifically above content of the invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention
The technology realized all belongs to the scope of the present invention.
Detailed description of the invention
Fig. 1：The agarose gel electrophoresis figure of lactobacillus plantarum PC518 Plasmid DNA is extracted with 7 kinds of schemes.
Fig. 2：7 kinds of schemes extract the concentration mensuration result of lactobacillus plantarum PC518 Plasmid DNA.
Fig. 3：By we modification method and gram-positive bacterium Plasmid Isolation Kit (Solarbio, Beijing, in
State) obtain Plasmid DNA comparison figure.A, the concentration of Plasmid DNA, the Plasmid DNA of B, agarose gel electrophoresis.Swimming lane 1 and 2：
The Plasmid DNA of lactobacillus plantarum PC518；Swimming lane 3 and 4：The Plasmid DNA of lactobacillus plantarum 9L15；Swimming lane 5 and 6：Cibarium Wei Si Shi
The Plasmid DNA of bacterium M2；Swimming lane 7 and 8：The Plasmid DNA of lactobacillus plantarum JS193；Swimming lane 9 and 10：The plasmid of lactobacillus plantarum 410
DNA.Plasmid DNA is by Solarbio gram-positive bacteria Plasmid Isolation Kit (odd number trace) or our modification method (even number
Road) it obtains.
Specific embodiment
The pretreatment reagent kit and method that embodiment 1, bacterial plasmid of the present invention extract
1, kit forms
Component A：100mg/ml lysozyme
B component：5% (referring to 5g/100ml) glucose solution
2, application method
Bacterium is taken, 100mg/ml lysozyme is added, then be incubated for 10 minutes at 37 DEG C, after bacteriolyze enzymatic treatment, by suspension
In 11000 × g, it is centrifuged 1 minute at room temperature；It washes twice again, steps are as follows：It will be deposited in 5% glucose solution of 500 μ l
It gently suspends, 11000 × g is centrifuged 1 minute, and supernatant is discarded.
The pretreatment reagent kit and method that embodiment 2, bacterial plasmid of the present invention extract
1, kit forms
Component A：120mg/ml lysozyme
B component：4% glucose solution
2, application method
Bacterium is taken, 150mg/ml lysozyme is added, then be incubated for 10 minutes at 37 DEG C, after bacteriolyze enzymatic treatment, by suspension
In 11000 × g, it is centrifuged 1 minute at room temperature；It washes twice again, steps are as follows：It will be deposited in 5% glucose solution of 500 μ l
It gently suspends, 11000 × g is centrifuged 1 minute, and supernatant is discarded.
The pretreatment reagent kit and method that embodiment 3, bacterial plasmid of the present invention extract
1, kit forms
Component A：160mg/ml lysozyme
B component：5% glucose solution
2, application method
Bacterium is taken, 200mg/ml lysozyme is added, then be incubated for 10 minutes at 37 DEG C, after bacteriolyze enzymatic treatment, by suspension
In 11000 × g, it is centrifuged 1 minute at room temperature；It washes twice again, steps are as follows：It will be deposited in 5% glucose solution of 500 μ l
It gently suspends, 11000 × g is centrifuged 1 minute, and supernatant is discarded.
The pretreatment reagent kit and method that embodiment 4, bacterial plasmid of the present invention extract
1, kit forms
Component A：80mg/ml lysozyme
B component：25mmol/L Tris-HCl (pH8.0), 10mmol/L EDTA, 50mmol/L glucose, 100 μ g/ml
RNase A
2, application method
Bacterium is taken, 100mg/ml lysozyme is added, then be incubated for 10 minutes at 37 DEG C, after bacteriolyze enzymatic treatment, by suspension
In 11000 × g, it is centrifuged 1 minute at room temperature；It washes twice again, steps are as follows：It will gently be hanged in the solution I for being deposited in 500 μ l
Floating, 11000 × g is centrifuged 1 minute, and supernatant is discarded.
Embodiment 5, bacterial plasmid extraction agent box of the present invention and method
1, kit forms
(1) pretreatment reagent kit
Any one kit that Examples 1 to 4 is recorded；
(2) lytic reagent box
Solution I：25mmol/L Tris-HCl (pH8.0), 10mmol/L EDTA, 50mmol/L glucose, 100 μ g/ml
RNase A
Solution II：250mmol/L NaOH, 1% (W/V) SDS (lauryl sodium sulfate)；
Solution III：3mol/L potassium acetate, 5mol/L acetic acid；
Solution IV：7.5 10mmol/L Tris-HCl of pH, 80% ethyl alcohol；
Solution V：10mmol/L Tris-HCl pH 7.5.
2, application method
(1) pre-treatment
Any one kit recorded using Examples 1 to 4 is carried out according to the corresponding method that Examples 1 to 4 is recorded
Processing；
(2) it cracks
1. 250 μ l solution II (ingredients are added in the thallus to suspend again to 250 μ l solution Is：250mmol/L NaOH,
1% (W/V) SDS (lauryl sodium sulfate)), leniently spinning upside down 6-8 times cracks thallus sufficiently.
2. 350 μ l solution III (ingredients are added into centrifuge tube：3mol/L potassium acetate, 5mol/L acetic acid), leniently immediately
It spins upside down 6-8 times, mixes well, will occur white flock precipitate at this time.13,400 × g is centrifuged 10min.
3. supernatant is transferred in adsorption column (adsorption column is put into collecting pipe), pay attention to trying not that precipitating is sucked out.
13,400 × g is centrifuged 30-60sec, outwells the waste liquid in collecting pipe, adsorption column is put into collecting pipe.
4. 500 μ l solution IV (ingredients are added into adsorption column：7.5 10mmol/L Tris-HCl of pH, 80% ethyl alcohol),
13,400 × g is centrifuged 30-60sec, outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe.
5. 50 μ l solution V (components are added into adsorption column：10mmol/L Tris-HCl pH 7.5), 13,400 × g from
Heart 1min, obtains plasmid solution.
Illustrate beneficial effects of the present invention below by way of the mode of test example.
Test example 1, bacterial plasmid method for extracting of the present invention
1 experimental strain
The lactobacillus plantarum PC518 that is isolated from Chinese pickle, 410,9L15 and JS193 bacterial strain.Cibarium Wei Si Salmonella
(Weissella cibaria) M2 bacterial strain is separated from giant panda enteron aisle.Under static anaerobic condition, bacterial strain exists
It is cultivated 20 hours for 37 DEG C in MRS culture medium.
Lactobacillus plantarum PC518,410,9L15 and JS193 bacterial strain, cibarium Wei Si Salmonella (Weissella cibaria) M2
Bacterial strain is Clinical isolation, is determined as lactobacillus plantarum and cibarium Wei Si Salmonella (Weissella by identification
cibaria)。
2 conditional filterings
2.1 screening technique
2.1.1 bacteriolyze enzymatic treatment and removal
This research shares 7 schemes.8 milliliters of culture solution (lactobacillus plantarums are harvested in sterile Eppordf pipe
PC518), 11000 × g is centrifuged 1 minute, and precipitating is extracted for plasmid.Bacteriolyze enzymatic treatment and removal scheme are as shown in table 1.
The bacteriolyze enzymatic treatment and removal scheme of 1. 7 lactobacillus plantarum PC518 thallus of table
a：The lysozyme of final concentration of 100mg/ml is added in solution I；b：Final concentration of 10mg/ml is added in solution I
Lysozyme；c：It is prepared in sterile water；d:250 μ l solution I ingredients.
In 250 μ l solution I (solution Is：The Portugal 25mmol/L Tris-HCl (pH8.0), 10mmol/L EDTA, 50mmol/L
Grape sugar, 100 μ g/ml RNase A) in 100mg/ml or 10mg/ml lysozyme is added, then be incubated for 10 at 37 DEG C or 50 DEG C
Minute or 30 minutes, after bacteriolyze enzymatic treatment, by suspension in 11000 × g, be centrifuged 1 minute at room temperature.Finally, in order to remove completely
Lysozyme is removed, is washed twice：It suspends 5% glucose solution for being deposited in 500 μ l or gently in solution I, 11000 × g centrifugation
1 minute, supernatant is discarded.
2.1.2 plasmid extracts
1. 250 μ l solution II (ingredients are added in the thallus to suspend again to 250 μ l solution Is：250mmol/L NaOH,
1% (W/V) SDS (lauryl sodium sulfate)), leniently spinning upside down 6-8 times cracks thallus sufficiently.
2. 350 μ l solution III (ingredients are added into centrifuge tube：3mol/L potassium acetate, 5mol/L acetic acid), leniently immediately
It spins upside down 6-8 times, mixes well, will occur white flock precipitate at this time.13,400 × g is centrifuged 10min.
3. supernatant is transferred in adsorption column (adsorption column is put into collecting pipe), pay attention to trying not that precipitating is sucked out.
13,400 × g is centrifuged 30-60sec, outwells the waste liquid in collecting pipe, adsorption column is put into collecting pipe.
4. 500 μ l solution IV (ingredients are added into adsorption column：7.5 10mmol/L Tris-HCl of pH, 80% ethyl alcohol),
13,400 × g is centrifuged 30-60sec, outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe.
5. 50 μ l solution V (components are added into adsorption column：10mmol/L Tris-HCl pH 7.5), 13,400 × g from
Heart 1min, obtains plasmid solution.
2.1.3 plasmid measures
Plasmid DNA is evaluated by horizontal electrophoresis system (U.S. Bio-Rad) agarose gel electrophoresis.The number of band
The quality and yield of extracted Plasmid DNA are represented with brightness.In addition, Plasmid DNA is by 2000 spectrophotometer of NanoDrop
(Thermo Scientific, the U.S.) it is quantitative at 260 and 280nm.The A260/A280 ratio of high-quality DNA is 1.8-2.0.
2.2 the selection result
Agarose gel electrophoresis results show that the quality for the Plasmid DNA extracted with P1, P2 and P3 scheme and yield are above
P4, P5, P6 and P7 scheme.It is obvious that having 8-9 identifiable bands, P4, P5 and the side P6 on the swimming lane of P1, P2 and P3 scheme
There was only 3-5 weak band on the swimming lane of case, and the band on the swimming lane of P7 scheme is disperse.In addition, in P1, P2 and P3 scheme
In swimming lane, the band of the swimming lane of P2 scheme be it is most bright, the band of the swimming lane of P1 scheme is most weak (Fig. 1).
The A260/A280 ratio for the Plasmid DNA that 7 kinds of schemes are extracted is between 1.8~2.0.The matter of P1, P2 and P3 scheme
The concentration of grain DNA is above the extracted plasmid of P4, P5 and P6 scheme.In addition to the extracted plasmid of P7 scheme, P2 scheme is extracted
Plasmid concentration highest, P5 scheme extract plasmid DNA concentration it is minimum (Fig. 2).The measurement result and agarose of spectrophotometer
Gel electrophoresis result is almost the same.
When lysozyme concentration for the treatment of is higher and lysozyme is removed before alkaline lysis step, the yield of Plasmid DNA and
Quality dramatically increases, such as has used 100mg/ml lysozyme and increased P1, P2 and the side P3 of lysozyme removal step
Case；If only improving lysozyme concentration for the treatment of as P5 scheme, good result would not be obtained.
Under 37 DEG C of bacteriolyze enzymatic treatments, the quality of the Plasmid DNA of P1, P2, P3, P4, P5 and P6 is superior to 50 DEG C of P7 schemes
Extracted Plasmid DNA (Fig. 1).Speculate that temperature increases the crisis of survival that will lead to cell, and generates self consumption phenomenon.P1 and
Unique difference of P2 scheme is the duration of bacteriolyze enzymatic treatment, the results showed that the extension of bacteriolyze enzymatic treatment is unfavorable for mentioning for plasmid
It takes.The lysozyme digestion time will lead to the loss (Cataloluk, 2003) of plasmid more than 20 minutes at 37 DEG C.Long lysozyme
Processing allows endogenous nucleases to become activity (Ledeboer et al., 1976).
The adjustable membrane permeability of washing solution containing 5% glucose, and intracellular content is controlled by osmotic balance
The release of object.The P3 of the washing solution of 5% glucose is free of than using using the P2 scheme of the washing solution containing 5% glucose
Scheme obtains better result.
Therefore, the present invention passes through 5% glucose or solution I washing removal after using 100mg/ml bacteriolyze enzymatic treatment
It after lysozyme, then carries out alkaline lysis processing and extracts plasmid, the plasmid DNA concentration that extracts is high, may be implemented effectively to extract
Purpose, currently preferred lysozyme processing mode are the mode of P2：100mg/ml is added in 250 μ l solution Is, then at 37 DEG C
It is lower to be incubated for 10 minutes, after bacteriolyze enzymatic treatment, by suspension in 11000 × g, it is centrifuged 1 minute at room temperature, in order to completely remove bacteriolyze
Enzyme washes twice：It will gently suspend in 5% glucose solution for being deposited in 500 μ l, 11000 × g is centrifuged 1 minute, by supernatant
Liquid discards.
3 commercial kits compare
3.1 method
Respectively with gram-positive bacteria Plasmid Isolation Kit (Solarbio, Beijing, China) and the preferred process of the present invention
(after handling in the way of P2 during 2.1.1 is saved, according still further to the processing of 2.1.2 section) extracts lactobacillus plantarum PC518,410,9L15
With the Plasmid DNA of JS193 bacterial strain and cibarium Wei Si Salmonella M2 bacterial strain.With agarose gel electrophoresis and spectrophotometric determination matter
Grain DNA.
3.2 result
Compared to gram-positive bacteria Plasmid Isolation Kit (Solarbio, Beijing, China), we have improved method
There is higher plasmid extraction efficiency.When we use modification method, the band on gel is more brighter, Fig. 3 B), plasmid is dense
Du Genggao (Fig. 3 A).Compared with commercial kits, 10.6,9.5,1.5,6 and have been respectively increased in the plasmid concentration of test strain
5.6 again.
The experiment results show that the present invention can effectively improve the extraction efficiency of bacterial plasmid, wherein to lactobacillus plantarum
Extraction effect is preferable.
To sum up, the present invention significantly improves plasmid extraction efficiency by specific pretreatment mode, and application prospect is excellent.
Claims (10)
1. a kind of pretreatment reagent kit of bacterial plasmid extracting, it is characterised in that：It includes following component：
Component A：80-160mg/ml lysozyme；
B component：Cleaning solution.
2. pretreatment reagent kit according to claim 1, it is characterised in that：The component A is 100mg/ml lysozyme.
3. pretreatment reagent kit according to claim 1, it is characterised in that：The cleaning solution is 4~6% (w/v) grapes
Sugar juice or solution I；The solution I is added with 10mmol/L EDTA, 50mmol/L glucose, 100 μ g/ml RNase
The Tris-HCl solution of A, wherein the concentration of Tris-HCl solution is 25mmol/L.
4. pretreatment reagent kit according to claim 3, it is characterised in that：The concentration of the glucose solution is 5%.
5. a kind of bacterial plasmid extraction agent box, it is characterised in that：It includes following component：
(1) pretreatment reagent kit described in Claims 1 to 4 any one；
(2) plasmid extraction kit.
6. extraction agent box according to claim 5, it is characterised in that：The component (two) is alkaline lysis method of extracting plasmid
Kit.
7. extraction agent box according to claim 6, it is characterised in that：The kit packet of the alkaline lysis method of extracting plasmid
Include following ingredient：
Solution I：Added with 10mmol/L EDTA, 50mmol/L glucose, 100 μ g/ml RNase A Tris-HCl solution,
Wherein, the concentration of Tris-HCl solution is 25mmol/L；
Solution II：Concentration added with 1% (W/V) SDS is the NaOH solution of 250mmol/L；
Solution III：The mixed solution of potassium acetate and acetic acid, the concentration 3mol/L of potassium acetate, the concentration of acetic acid are 5mol/L；
Solution IV：Every 1L contains 10mmol Tris-HCl and 0.80L ethyl alcohol, remaining is water；
Solution V：The Tris-HCl solution of 10mmol/L.
8. a kind of pre-treating method of bacterial plasmid extracting, it is characterised in that：It is using Claims 1 to 4 any one institute
The pretreatment reagent kit processing stated, steps are as follows：Microorganism is handled with component A, is then washed with B component.
9. a kind of bacterial plasmid method for extracting, it is characterised in that：It is using reagent described in claim 5~7 any one
Box extracting, steps are as follows：Pre-treatment is carried out according to claim 8 the method, reuses plasmid extraction kit extraction.
10. method for extracting according to claim 9, it is characterised in that：The plasmid extraction kit be claim 6 or
Plasmid extraction kit in 7.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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Cited By (2)
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| 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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