CN115369112A - Binding liquid for endotoxin-removing plasmid extraction, kit and plasmid extraction method - Google Patents
Binding liquid for endotoxin-removing plasmid extraction, kit and plasmid extraction method Download PDFInfo
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Abstract
The invention relates to the field of plasmid extraction, and particularly discloses a binding solution for endotoxin-removing plasmid extraction, a kit and a plasmid extraction method. A binding solution for endotoxin-removing plasmid extraction comprises a solvent, guanidine isothiocyanate and triton X-114, wherein the molar concentration of the guanidine isothiocyanate in the binding solution is 2-5mol/L, and the volume percentage of the triton X-114 in the binding solution is 1-5% (V/V). The invention has the advantages of improving the efficiency of extracting plasmids and improving the effect of removing endotoxin.
Description
Technical Field
The invention relates to the field of plasmid extraction, in particular to binding solution and kit for endotoxin-removing plasmid extraction and a plasmid extraction method.
Background
Plasmids are extrachromosomal, independently heritable, covalently closed double-stranded DNA molecules that exist in bacteria and other organisms and which provide host cells with phenotypes such as resistance to drugs and the ability to break down complex organic matter. The wild plasmid can be used as a vector of genetic engineering after being modified, and the plasmid vector has extremely wide application value in the genetic engineering. Therefore, the isolation and extraction of plasmids is one of the most common and basic experimental techniques in molecular biology.
Bacterial plasmids are the most commonly used genetic engineering vectors, and plasmids extracted by a HiPure Plasmid kit can be directly used for automatic sequencing, enzyme digestion, PCR, labeling and the like. However, we are susceptible to various contaminations during plasmid extraction, and the most problematic of them is endotoxin.
Endotoxin, also known as lipopolysaccharide or LPS, is a component of gram-negative (e.g. e.coli) cell membranes. The outer lipid component of the bacterial outer membrane is composed entirely of endotoxin molecules. Coli contains about 2 million LPS molecules, each consisting of hydrophobic lipid a, a complex polysaccharide chain, and negatively charged phosphate groups. Each endotoxin thus contains both hydrophobic and hydrophilic and charged regions, giving it the unique property of interacting with other molecules. Bacteria have a low endotoxin content on their surface when they are actively growing, and release a large amount of endotoxin once they die. During lysis in plasmid extraction, endotoxins are released from the outer membrane of the bacteria into the lysate.
The chemical structure, properties and tendency to form micelle structures of endotoxin molecules often co-exist with plasmid DNA and are co-purified.
Endotoxin can have a severe effect on DNA transfection of primary cells and also on sensitivity to cultured cells, and increased levels of endotoxin can lead to a dramatic decrease in transfection efficiency. Furthermore, the use of endotoxin-free plasmid DNA is of particular importance for gene therapy applications, since endotoxins cause fever, endotoxic shock and can activate the complement cascade in animals and humans. Endotoxin also interferes with transfected immune cells in vitro, such as macrophages and B cells, because it can non-specifically elicit an immune response. The response involves the synthesis of immune transmitters such as IL-1 and prostaglandins. In order to prevent erroneous judgment of the experimental results, plastic products, culture media, serum and plasmid DNA without endotoxin contamination must be used in the experiment.
As a conventional method for removing endotoxin, there is a liquid phase separation method in which sodium deoxycholate is bound to the lipid fraction of endotoxin by using a detergent such as Triton X-114 (Triton X-114), and the endotoxin is extracted by the liquid phase separation method to obtain an endotoxin-free supernatant, which is then subjected to column adsorption to further purify the plasmid.
However, when endotoxin is removed by an extraction method, the extraction speed is slow, and the phenomenon of unobvious layering is easy to occur, so that the whole process period is long, the plasmid extraction efficiency is low, and the endotoxin removal effect is general, so that the improvement space is provided.
Disclosure of Invention
In order to improve the efficiency of extracting plasmids and improve the effect of removing endotoxin, the application provides a binding solution for removing endotoxin plasmids, a kit and a plasmid extraction method.
In a first aspect, the application provides a binding solution for endotoxin removal plasmid extraction, which adopts the following technical scheme:
a binding solution for endotoxin-removing plasmid extraction comprises a solvent, guanidine isothiocyanate and triton X-114, wherein the molar concentration of the guanidine isothiocyanate in the binding solution is 2-5mol/L, and the volume percentage of the triton X-114 in the binding solution is 1-5% (V/V).
Preferably, the molar concentration of the guanidinium isothiocyanate in the binding solution is 3-4mol/L, and the volume percentage of the triton X-114 in the binding solution is 2-4% (V/V).
Preferably, the molar concentration of the guanidinium isothiocyanate in the binding solution can be 2-3mol/L, 3-3.5mol/L, 3.5-4mol/L and 4-5mol/L, and the volume percentage of the triton X-114 in the binding solution can be 1-2% (V/V), 2-3% (V/V), 3-4% (V/V) and 4-5% (V/V).
By adopting the technical scheme, through the compounding of the triton-114 and the guanidinium isothiocyanate in a specific concentration range, the triton-114 and the guanidinium isothiocyanate can be coupled, and the endotoxin can be removed by adding the binding solution in the process of extracting the plasmid, so that the step of extracting the plasmid and then passing through a column in the prior art is omitted, the plasmid can be extracted more efficiently and rapidly, the problem of unobvious layering does not exist, and the effect of removing the endotoxin is better and more stable.
Moreover, through the compounding of the triton-114 and the guanidinium isothiocyanate in a specific concentration range, the yield of plasmids can not be lost when the endotoxin is removed, so that the yield of the plasmids is higher.
Preferably, the solvent is water.
By adopting the technical scheme, water is used as a solvent, the dissolving effect of the triton-114 and the guanidinium isothiocyanate is better, so that the triton-114 and the guanidinium isothiocyanate can better react with each other to realize coupling, the endotoxin removing effect of the obtained binding solution is better, the yield of plasmids is not easy to lose, and the yield of the plasmids is ensured while the endotoxin is better removed.
In a second aspect, the present application provides an endotoxin plasmid extraction kit, which adopts the following technical scheme:
an endotoxin-removing plasmid extraction kit at least comprises the binding solution.
Through adopting above-mentioned technical scheme, when adopting to remove endotoxin plasmid extraction kit to extract the plasmid, need not the extraction step, can extract and obtain the plasmid of removing endotoxin, extraction efficiency is higher, and endotoxin gets rid of the effect preferred and comparatively stable.
Preferably, the endotoxin-removing plasmid extraction kit further comprises a bacterial body weight suspension, a bacterial lysate, a neutralization solution, a deproteinization washing solution, a desalination washing solution and an eluent.
By adopting the technical scheme, the plasmid extraction effect is better through the matching of the bacterial body weight suspension, the bacterial lysate, the neutralization solution, the deproteinized washing solution, the desalted washing solution and the eluent.
In a third aspect, the present application provides a method for extracting endotoxin-free plasmid, which adopts the following technical scheme:
a method for extracting endotoxin-removing plasmid comprises the following steps:
step 1), mixing the thalli and the thalli heavy suspension to obtain a bacterium heavy suspension;
step 2), adding the thallus lysate into the bacteria resuspension, and uniformly mixing to obtain a bacteria treatment solution;
step 3), adding the neutralization solution into the bacterial treatment solution, uniformly mixing, and taking supernatant;
step 4), adding the combined liquid into the supernatant, and uniformly mixing to obtain a mixed liquid;
step 5), transferring the mixed solution to an adsorption column, carrying out centrifugal filtration, and discarding filtrate;
step 6), adding a deproteinized washing solution into an adsorption column, carrying out centrifugal filtration, and discarding the filtrate;
step 7), adding a desalting washing solution into an adsorption column, carrying out centrifugal filtration, and discarding filtrate;
step 8), removing ethanol in the adsorption column;
step 9), adding eluent into the adsorption column, and carrying out centrifugal elution to obtain endotoxin-removed plasmids;
the binding solution is the binding solution.
By adopting the technical scheme, the combined liquid is added into the supernatant without extraction, so that the endotoxin can be removed, the efficiency of extracting the plasmid is high, the effect of removing the endotoxin is good, and the plasmid yield is high.
Preferably, in the step 4), the volume ratio of the binding solution to the supernatant is 1:1.
through adopting above-mentioned technical scheme, through the volume ratio of specifically selecting the binding solution and supernatant for get rid of endotoxin's effect preferred, reduce the too much lower condition that leads to post adsorption efficiency of binding solution simultaneously, efficiency is higher, and the effect is better.
Preferably, in the step 5), the maximum relative centrifugal force is 13000g and the centrifugation is 30-60s during centrifugal filtration.
By adopting the technical scheme and adopting a specific process for centrifugation, the plasmid and the filtrate are separated more thoroughly, and the quality of the extracted plasmid is better.
In summary, the present application has the following beneficial effects:
1. because this application is through the compound of triton-114 and guanidinium isothiocyanate in specific concentration range, triton-114 and guanidinium isothiocyanate can couple, through adding the binding liquid in the process of extracting the plasmid, can realize that the endotoxin is got rid of, has exempted from among the prior art earlier the step of extraction post again for the plasmid is drawed and can be high-efficient quick more, and does not have the obscure problem of layering, gets rid of the effect of endotoxin better and comparatively stable, plasmid output is higher.
2. Preferentially, through adopting water as the solvent in this application, the dissolving effect of triton-114 and guanidinium isothiocyanate is better for triton-114 and guanidinium isothiocyanate can better interact to realize coupling, the effect of endotoxin is got rid of to the binding solution that obtains is better, and the output of difficult loss plasmid guarantees the plasmid output when getting rid of endotoxin better.
3. According to the method, the combined liquid is added into the supernate, the endotoxin can be removed without extraction, the efficiency of extracting the plasmid is high, the effect of removing the endotoxin is good, and the plasmid yield is high.
Detailed Description
The present application will be described in further detail with reference to examples.
Example 1
A binding solution for endotoxin-removing plasmid extraction comprises the following components:
solvent, guanidine isothiocyanate and triton X-114.
Wherein the solvent is water.
Guanidinium isothiocyanate is commercially available from CAS:593-84-0.
Triton X-114 was obtained from a commercial source, CAS:9036-19-5.
The preparation method of the binding solution for endotoxin-removing plasmid extraction comprises the following steps:
236.3g of guanidinium isothiocyanate is weighed and added into 800ml of pure water, the mixture is stirred until the guanidinium isothiocyanate is completely dissolved to obtain a guanidinium isothiocyanate solution, then 10ml of triton X-114 is weighed and added into the guanidinium isothiocyanate solution, the mixture is stirred uniformly to obtain a mixed solution, then the mixed solution is injected into a 1L volumetric flask, then the pure water is added to the volumetric flask to reach a constant volume of 1L, and the combined solution for endotoxin-removed plasmid extraction is obtained.
Example 2
A binding solution for endotoxin-removing plasmid extraction comprises the following components:
solvent, guanidinium isothiocyanate, triton X-114.
Wherein the solvent is water.
Guanidinium isothiocyanate was obtained commercially from CAS:593-84-0.
Triton X-114 was obtained from a commercial source, CAS:9036-19-5.
The preparation method of the binding solution for endotoxin-removing plasmid extraction comprises the following steps:
354.5g of guanidinium isothiocyanate is weighed and added into 800ml of pure water, the mixture is stirred until the guanidinium isothiocyanate is completely dissolved to obtain a guanidinium isothiocyanate solution, then 20ml of triton X-114 is weighed and added into the guanidinium isothiocyanate solution, the mixture is stirred uniformly to obtain a mixed solution, then the mixed solution is injected into a 1L volumetric flask, then pure water is added to the volumetric flask to a constant volume of 1L, and the binding solution for extracting the endotoxin-removed plasmid is obtained.
Example 3
A binding solution for endotoxin-removing plasmid extraction comprises the following components:
solvent, guanidine isothiocyanate and triton X-114.
Wherein the solvent is water.
Guanidinium isothiocyanate was obtained commercially from CAS:593-84-0.
Triton X-114 was obtained from a commercial source, CAS:9036-19-5.
The preparation method of the binding solution for endotoxin-removing plasmid extraction comprises the following steps:
413.6g of guanidinium isothiocyanate is weighed and added into 800ml of pure water, the mixture is stirred until the guanidinium isothiocyanate is completely dissolved to obtain a guanidinium isothiocyanate solution, then 30ml of triton X-114 is weighed and added into the guanidinium isothiocyanate solution, the mixture is stirred uniformly to obtain a mixed solution, then the mixed solution is injected into a 1L volumetric flask, then the pure water is added to the volumetric flask to reach the constant volume of 1L, and the combined solution for endotoxin-removed plasmid extraction is obtained.
Example 4
A binding solution for extracting endotoxin-removing plasmids comprises the following components:
solvent, guanidine isothiocyanate and triton X-114.
Wherein the solvent is water.
Guanidinium isothiocyanate is commercially available from CAS:593-84-0.
Triton X-114 was obtained from a commercial source, CAS:9036-19-5.
The preparation method of the binding solution for endotoxin-removing plasmid extraction comprises the following steps:
weighing 472.6g of guanidinium isothiocyanate, adding the guanidinium isothiocyanate into 800ml of pure water, stirring until the guanidinium isothiocyanate is completely dissolved to obtain a guanidinium isothiocyanate solution, then weighing 40ml of triton X-114, adding the triton X-114 into the guanidinium isothiocyanate solution, uniformly stirring to obtain a mixed solution, then injecting the mixed solution into a 1L volumetric flask, and then adding the pure water to a constant volume of 1L to obtain a binding solution for endotoxin-removed plasmid extraction.
Example 5
A binding solution for extracting endotoxin-removing plasmids comprises the following components:
solvent, guanidine isothiocyanate and triton X-114.
Wherein the solvent is water.
Guanidinium isothiocyanate was obtained commercially from CAS:593-84-0.
Triton X-114 was obtained from a commercial source, CAS:9036-19-5.
The preparation method of the binding solution for endotoxin-removing plasmid extraction comprises the following steps:
590.8g of guanidinium isothiocyanate is weighed and added into 800ml of pure water, the mixture is stirred until the guanidinium isothiocyanate is completely dissolved to obtain a guanidinium isothiocyanate solution, then 50ml of triton X-114 is weighed and added into the guanidinium isothiocyanate solution, the mixture is stirred uniformly to obtain a mixed solution, then the mixed solution is injected into a 1L volumetric flask, then pure water is added to the volumetric flask to a constant volume of 1L, and the binding solution for extracting the endotoxin-removed plasmid is obtained.
Application example 1
A kit for extracting endotoxin-removed plasmid comprises a bacterial body suspension, a bacterial lysate, a neutralization solution, a binding solution, a deproteinized washing solution, a desalted washing solution and an eluent.
The preparation method of the bacterial weight suspension comprises the following steps:
weighing 2.42g of tris (hydroxymethyl) aminomethane, 9.91g of glucose and 3.72g of ethylenediamine tetraacetic acid respectively, adding tris (hydroxymethyl) aminomethane, glucose and ethylenediamine tetraacetic acid into 900ml of pure water, stirring until the materials are completely dissolved, adding hydrochloric acid to adjust the pH value to 8.0 to obtain a mixed solution, injecting the mixed solution into a 1L volumetric flask, and adding pure water to a constant volume of 1L to obtain a bacterial suspension.
The preparation method of the thallus lysate comprises the following steps:
respectively weighing 8g of sodium hydroxide and 10g of sodium dodecyl sulfate, adding the sodium hydroxide and the sodium dodecyl sulfate into 900ml of pure water, stirring until the sodium hydroxide and the sodium dodecyl sulfate are completely dissolved to obtain a mixed solution, injecting the mixed solution into a 1L volumetric flask, and adding the pure water to a constant volume of 1L to obtain a thallus lysate.
The preparation method of the neutralization solution comprises the following steps:
196.3g of potassium acetate is weighed and added into 600ml of pure water, the mixture is stirred until the potassium acetate is completely dissolved to obtain a potassium acetate solution, then 100ml of acetic acid is measured by a measuring cylinder and added into the potassium acetate solution, the mixture is uniformly mixed to obtain a mixed solution, the mixed solution is injected into a 1L volumetric flask, and then pure water is added to the volumetric flask to reach a constant volume of 1L, so that a neutralizing solution is obtained.
The deproteinizing washing solution is prepared as follows:
weighing 382g of guanidine hydrochloride and 6g of tris (hydroxymethyl) aminomethane respectively, adding the guanidine hydrochloride and the tris (hydroxymethyl) aminomethane into 100ml of pure water, stirring until the guanidine hydrochloride and the tris (hydroxymethyl) aminomethane are completely dissolved to obtain a premixed solution, then adding hydrochloric acid into the premixed solution to adjust the pH to 5.5, measuring 300ml of isopropanol with the concentration of 30% by using a measuring cylinder, adding the isopropanol into the premixed solution, uniformly mixing to obtain a mixed solution, injecting the mixed solution into a 1L volumetric flask, and adding pure water to a constant volume of 1L to obtain the deproteinized washing solution.
The desalting washing solution was prepared as follows:
2.4g of tris (hydroxymethyl) aminomethane and 10g of sodium chloride are respectively weighed, the tris (hydroxymethyl) aminomethane and the sodium chloride are added into 500ml of pure water, stirred until completely dissolved, then injected into a 1L volumetric flask, and then added with the pure water to a constant volume of 1L.
The eluent was prepared as follows:
weighing 1.2g of tris (hydroxymethyl) aminomethane, adding tris (hydroxymethyl) aminomethane into 900ml of pure water, stirring until the tris (hydroxymethyl) aminomethane is completely dissolved, adding hydrochloric acid to adjust the pH value to 8.5, injecting into a 1L volumetric flask, and adding pure water to a constant volume of 1L.
The binding solution was the binding solution of example 1.
Application example 2
An endotoxin-removing plasmid extraction kit, which is different from the application example 1 only in that:
the binding solution was the binding solution of example 2.
Application example 3
An endotoxin-removing plasmid extraction kit, which is different from the application example 1 only in that:
the binding solution was the binding solution of example 3.
Application example 4
An endotoxin-removing plasmid extraction kit, which is different from the application example 1 only in that:
the binding solution was the binding solution of example 4.
Application example 5
An endotoxin-removing plasmid extraction kit, which is different from the application example 1 only in that:
the binding solution was the binding solution of example 5.
Experimental example 1
A method for extracting endotoxin-removed plasmids by using an endotoxin-removed plasmid extraction kit comprises the following steps:
step 1), mixing the thalli and the thalli heavy suspension to obtain a bacterium heavy suspension, which comprises the following specific steps:
step 1-1), adding 0.5ml of bacterial weight suspension into the RNase A dry powder, sucking, beating and uniformly mixing for 10 times to fully dissolve the RNase A dry powder, and storing at 2 ℃ to obtain a bacterial weight suspension/RNase A mixed solution.
Step 1-2), inoculating Escherichia coli E.coli containing plasmid into a culture flask containing 15ml of ampicillin culture solution, and performing shake culture at 37 ℃ for 16 hours to amplify the plasmid. Detecting the bacteria content to reach OD600 of 0.8 to obtain the bacteria culture solution.
Step 1-3), centrifuging the bacterial culture solution for 10 minutes under the condition that the maximum relative centrifugal force is 5000g, and collecting 10ml of thallus.
Step 1-4), the culture medium is discarded, and the residual liquid is sucked up by gently flapping on absorbent paper. Then putting the thalli into a test tube, then adding 500 mu l of mixed liquid of the thalli heavy suspension liquid/RNase A prepared in the step 1-1) into the test tube, and then putting the test tube into a vortex instrument for vortex to ensure that the bacteria are fully suspended until no bacterial lumps are seen, thus obtaining the bacterial heavy suspension.
Step 2), adding the thallus lysate into the bacteria resuspension, and uniformly mixing to obtain a bacteria treatment solution, which comprises the following specific steps:
adding 500 μ l thallus lysate into the bacteria heavy suspension, reversing and mixing for 10 times, standing at room temperature for 3min, and reversing and mixing for 1 time every 1min during the standing process to obtain the bacteria treatment solution.
And 3), adding the neutralization solution into the bacterial treatment solution, uniformly mixing, and taking supernatant, wherein the method specifically comprises the following steps:
step 3-1), 250. Mu.l of a neutralizing solution was added to the bacterial treatment solution, and immediately reversed 15 times to mix well, and flocculent precipitates were generated in the test tube and all flocculent precipitates were uniform white.
And 3-2), placing the test tube into a centrifugal machine, centrifuging for 10 minutes under the condition that the maximum relative centrifugal force is 13000g, and taking supernate.
And 4), adding the combined solution into the supernatant, and uniformly mixing to obtain a mixed solution, wherein the mixed solution comprises the following components:
and (3) transferring 0.95ml of the supernatant obtained in the step (3-2) to a centrifuge tube, then injecting 0.95ml of the binding solution into the supernatant, and reversing and uniformly mixing for 5 times to obtain a mixed solution.
And 5), transferring the mixed solution to an adsorption column, performing centrifugal filtration, and discarding filtrate, wherein the method comprises the following steps:
the HiPure DNA Mini Column III was mounted in a collection tube and 750. Mu.l of the mixture from step 4) was transferred to the Column. Centrifuging for 30s under the condition that the maximum relative centrifugal force is 13000g, discarding the filtrate, sleeving the column back to the collecting pipe, transferring 750 ul of the mixed solution prepared in the step 4) to the column, centrifuging for 30s under the condition that the maximum relative centrifugal force is 13000g, discarding the filtrate, sleeving the column back to the collecting pipe, transferring the rest of the mixed solution to the column, centrifuging for 30s under the condition that the maximum relative centrifugal force is 13000g, and discarding the filtrate.
Step 6), adding a deproteinizing washing solution into an adsorption column, performing centrifugal filtration, and discarding filtrate, wherein the details are as follows:
the column was returned to the collection tube, 600. Mu.l of deproteinizing washing solution was added to the column, and the column was centrifuged for 30 seconds under a condition of a maximum relative centrifugal force of 13000g, and the filtrate was discarded.
Step 7), adding a desalting washing solution into an adsorption column, performing centrifugal filtration, and discarding filtrate, wherein the details are as follows:
the column was returned to the collection tube, 600. Mu.l of desalting wash solution was added to the column, centrifuged for 30 seconds under conditions of a maximum relative centrifugal force of 13000g, and the filtrate was discarded.
And 8) removing the ethanol in the adsorption column, wherein the method comprises the following steps:
the column was pulled back into the collection tube and centrifuged for 2min at 13000g maximum relative centrifugal force to dry the column to remove ethanol.
Step 9), adding an eluent into the adsorption column, and carrying out centrifugal elution to obtain the plasmid without endotoxin, wherein the specific steps are as follows:
the column was fitted in a sterilized 1.5ml centrifuge tube, 80ul of eluent was added to the center of the membrane of the column, left to stand for 2min, centrifuged for 1min under a maximum relative centrifugal force of 13000g, the plasmids were eluted to give endotoxin-free plasmids, which were stored at-20 ℃.
The endotoxin-free plasmid extraction kit is the endotoxin-free plasmid extraction kit of application example 1.
Plasmid extraction experiments of high copy escherichia coli e.coli and low copy escherichia coli e.coli were performed according to the method of this experimental example, respectively.
Coli, a pkD large intestine knockout plasmid purchased from shanghai grain noon biotechnology limited, part of the information of the large intestine knockout plasmids nos. P1247 and pkD, are as follows:
a promoter: araBAD promoter.
Replicon: pSC101ori.
A terminator: λ tL3 terminator.
Prokaryotic resistance: ampicillin Amp.
Cloning the strain: escherichia coli DH5 alpha.
The culture conditions are as follows: LB medium, 30 ℃.
Expression host: escherichia coli.
The induction mode is as follows: arabinose.
Coli, part of the information on DH5 α (containing pUC18 plasmid) e.coli, which is purchased from shanghai junior biotechnology limited, under the designation LZJ6620, and DH5 α (containing pUC18 plasmid) e.coli is as follows:
the species are as follows: escherichia coli DH 5. Alpha. (pUC 18).
Culture medium: LB medium, containing 50. Mu.g/mL ampicillin sodium.
Growth conditions are as follows: aerobic at 37 ℃.
Experimental example 2
Compared with the experimental example 1, the method for extracting endotoxin-removed plasmids by using the endotoxin-removed plasmid extraction kit only has the following differences:
the endotoxin-free plasmid extraction kit is the endotoxin-free plasmid extraction kit of application example 2.
Experimental example 3
Compared with the experimental example 1, the method for extracting endotoxin-removed plasmids by using the endotoxin-removed plasmid extraction kit only has the following differences:
the endotoxin-free plasmid extraction kit is the endotoxin-free plasmid extraction kit of application example 3.
Experimental example 4
Compared with the experimental example 1, the method for extracting endotoxin-removed plasmids by using the endotoxin-removed plasmid extraction kit only has the following differences:
the endotoxin-free plasmid extraction kit is the endotoxin-free plasmid extraction kit of application example 4.
Experimental example 5
Compared with the experimental example 1, the method for extracting endotoxin-removed plasmids by using the endotoxin-removed plasmid extraction kit only has the following differences:
the endotoxin-free plasmid extraction kit is the endotoxin-free plasmid extraction kit of application example 5.
Comparative example 1
A binding solution for endotoxin-free plasmid extraction, which is different from example 3 only in that:
guanidine hydrochloride is used in place of guanidine isothiocyanate in equal amount.
Comparative example 2
A binding solution for endotoxin-free plasmid extraction, which is different from example 3 only in that:
the same amount of triton X-100 is adopted to replace triton X-114.
Comparative example 3
A binding solution for endotoxin-removing plasmid extraction comprises the following components:
solvent, guanidine isothiocyanate and triton X-114.
Wherein the solvent is water.
Guanidinium isothiocyanate was obtained commercially from CAS:593-84-0.
Triton X-114 was obtained from a commercial source, CAS:9036-19-5.
The preparation method of the binding solution for endotoxin-removing plasmid extraction comprises the following steps:
177.2g of guanidinium isothiocyanate is weighed and added into 800ml of pure water, the mixture is stirred until the guanidinium isothiocyanate is completely dissolved to obtain a guanidinium isothiocyanate solution, then 55ml of triton X-114 is measured and added into the guanidinium isothiocyanate solution, the mixture is stirred uniformly to obtain a mixed solution, then the mixed solution is injected into a 1L volumetric flask, and then the pure water is added to the volumetric flask to reach a constant volume of 1L, so that the combined solution for endotoxin-removed plasmid extraction is obtained.
Comparative example 4
A binding solution for extracting endotoxin-removing plasmids comprises the following components:
solvent, guanidinium isothiocyanate, triton X-114.
Wherein the solvent is water.
Guanidinium isothiocyanate is commercially available from CAS:593-84-0.
Triton X-114 was obtained from a commercial source, CAS:9036-19-5.
The preparation method of the binding solution for endotoxin-removing plasmid extraction comprises the following steps:
649.9g of guanidinium isothiocyanate is weighed and added into 800ml of pure water, the mixture is stirred until the guanidinium isothiocyanate is completely dissolved to obtain a guanidinium isothiocyanate solution, then 5ml of triton X-114 is weighed and added into the guanidinium isothiocyanate solution, the mixture is stirred uniformly to obtain a mixed solution, then the mixed solution is injected into a 1L volumetric flask, then pure water is added to the volumetric flask to be constant in volume to 1L, and the binding solution for endotoxin-free plasmid extraction is obtained.
Comparative application example 1
An endotoxin-removing plasmid extraction kit, which is different from the application example 1 only in that:
the binding liquid was the binding liquid of comparative example 1.
Comparative application example 2
An endotoxin-removing plasmid extraction kit, which is different from the application example 1 only in that:
the binding liquid was the binding liquid of comparative example 2.
Comparative application example 3
An endotoxin-removing plasmid extraction kit, which is different from the application example 1 only in that:
the binding liquid was the binding liquid of comparative example 3.
Comparative application example 4
An endotoxin-removing plasmid extraction kit, which is different from the application example 1 only in that:
the binding liquid was the binding liquid of comparative example 4.
Comparative experiment example 1
Compared with the experimental example 1, the method for extracting endotoxin-removed plasmids by using the endotoxin-removed plasmid extraction kit only has the following differences:
the endotoxin-removed plasmid extraction kit is the endotoxin-removed plasmid extraction kit of comparative application example 1.
Comparative experiment example 2
Compared with the experimental example 1, the method for extracting endotoxin-removed plasmids by using the endotoxin-removed plasmid extraction kit only has the following differences:
the endotoxin-removing plasmid extraction kit is the endotoxin-removing plasmid extraction kit of comparative application example 2.
Comparative experiment example 3
Compared with the experimental example 1, the method for extracting endotoxin-removed plasmids by using the endotoxin-removed plasmid extraction kit only has the following differences:
the endotoxin-removed plasmid extraction kit is the endotoxin-removed plasmid extraction kit of comparative application example 3.
Comparative experiment example 4
Compared with the experimental example 1, the method for extracting endotoxin-removed plasmids by using the endotoxin-removed plasmid extraction kit only has the following differences:
the endotoxin-removed plasmid extraction kit is the endotoxin-removed plasmid extraction kit of comparative application example 4.
Experiment 1
The plasmid yields of high copy e.coli and low copy e.coli of each experimental example and comparative experimental example were examined using a siermer fly NanoDrop spectrophotometer.
The specific experimental data for experiment 1 are detailed in table 1.
Experiment 2
The high copy E.coli, low copy E.coli plasmid of each experimental example and comparative experimental example were tested for endotoxin.
1. Preparation of bacterial endotoxin solution:
preparation of limulus reagent: 1.7ml of bacterial endotoxin water was added to the limulus reagent.
Preparing a color development matrix: 1.7ml of bacterial endotoxin water was added to the chromogenic substrate.
Preparation of azo reagent 1: the diazotization reagent 1 is added with 9.2ml of bacterial endotoxin water and 800ul 1 hydrochloric acid.
Preparation of an azo reagent 2: the azotization reagent 2 is added with 10ml of bacterial endotoxin water.
Preparing an azotizing agent 3: diazotizing reagent 3 was added with 10ml bacterial endotoxin water.
2. Bacterial endotoxin test methods:
preparing a sample: 1ul of plasmid +1ml of bacterial endotoxin water prepared in each experimental example and comparative experimental example, and vortexed uniformly.
25ul of the sample was added to 25ul of limulus reagent.
All the above reagents were incubated at 37 ℃ in the dark for 7min.
After warm bath, 25ul of color development matrix is added, vortex uniformly, and warm bath is carried out at 37 ℃ in the dark for 5min.
After the warm bath, 125ul of azotized reagent 1 was added and vortexed uniformly.
Then 125ul of azotization reagent 2 is added, and vortex mixing is carried out.
Then 125ul of azotized reagent 3 was added and vortexed uniformly.
The endotoxin concentration of the plasmid was calculated by observing the position of the plasmid shown on a standard curve of 0EU,0.1EU,0.5EU, 1EU.
The specific assay data for experiment 2 is detailed in table 2.
TABLE 2
According to the data in tables 1 and 2, when the binding solution contains the combination of guanidinium isothiocyanate and triton X-114, and guanidinium isothiocyanate and triton X-114 are combined under a specific concentration, the effect of removing endotoxin is better, and the yield of plasmids is higher, while when guanidinium isothiocyanate is replaced by other guanidinium salts or triton X-114 is replaced by other triton, the effect of removing endotoxin is obviously reduced, and the yield of plasmids is greatly reduced, so that the cost of extracting plasmids is greatly improved, and the economic value is poorer.
When guanidine isothiocyanate and triton X-114 are compounded, but guanidine isothiocyanate and triton X-114 are not compounded under a specific concentration, the effect of removing endotoxin is better than that of changing the raw materials of a compound system, but still is obviously lower than that of removing endotoxin when guanidine isothiocyanate and triton X-114 are compounded under a specific concentration, and meanwhile, the yield of plasmids is higher than that of changing the raw materials of the compound system, but still is obviously lower than that of plasmids when guanidine isothiocyanate and triton X-114 are compounded under a specific concentration.
In conclusion, guanidine isothiocyanate and triton X-114 are matched under specific concentration, so that a special coupling effect can be generated, and the effect of removing endotoxin without losing the yield of plasmids is realized.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.
Claims (8)
1. A binding solution for endotoxin-removing plasmid extraction is characterized in that: the solvent, guanidine isothiocyanate and triton X-114 are included, the molar concentration of the guanidine isothiocyanate in the binding solution is 2-5mol/L, and the volume percentage of the triton X-114 in the binding solution is 1-5% (V/V).
2. The binding solution for endotoxin-removing plasmid extraction according to claim 1, wherein: the molar concentration of the guanidinium isothiocyanate in the binding solution is 3-4mol/L, and the volume percentage of the triton X-114 in the binding solution is 2-4% (V/V).
3. The binding solution for endotoxin-free plasmid extraction according to claim 1or 2, wherein: the solvent is water.
4. A remove endotoxin plasmid extraction kit which characterized in that: the endotoxin-removing plasmid extraction kit comprising at least the binding solution according to any one of claims 1 to 3.
5. The endotoxin-free plasmid extraction kit as claimed in claim 4, wherein: the endotoxin-removing plasmid extraction kit also comprises a thallus suspension, a thallus lysate, a neutralization solution, a deproteinizing washing solution, a desalting washing solution and an eluent.
6. A method for extracting endotoxin-removing plasmids is characterized by comprising the following steps: the method comprises the following steps:
step 1), mixing the thalli with a thalli heavy suspension to obtain a bacterium heavy suspension;
step 2), adding the thallus lysate into the bacterial resuspension, and mixing uniformly to obtain a bacterial treatment solution;
step 3), adding the neutralization solution into the bacterial treatment solution, uniformly mixing, and taking supernatant;
step 4), adding the combined liquid into the supernatant, and uniformly mixing to obtain a mixed liquid;
step 5), transferring the mixed solution to an adsorption column, carrying out centrifugal filtration, and discarding filtrate;
step 6), adding a deproteinized washing solution into an adsorption column, carrying out centrifugal filtration, and discarding the filtrate;
step 7), adding a desalting washing solution into an adsorption column, carrying out centrifugal filtration, and discarding filtrate;
step 8), removing ethanol in the adsorption column;
step 9), adding eluent into the adsorption column, and carrying out centrifugal elution to obtain endotoxin-removed plasmids;
the binding solution according to any one of claims 1 to 3.
7. The method for extracting endotoxin-free plasmid of claim 6, wherein: in the step 4), the volume ratio of the binding solution to the supernatant is 1:1.
8. a method of extracting endotoxin-removing plasmid as claimed in claim 7, wherein: in the step 5), the maximum relative centrifugal force is 13000g during centrifugal filtration, and the centrifugation is 30-60s.
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| CN116162619A (en) * | 2023-04-26 | 2023-05-26 | 南京欧凯生物科技有限公司 | Low endotoxin plasmid extraction kit and extraction method thereof |
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