CN117535283B - Kit for extracting blood genome DNA by magnetic bead method and application thereof - Google Patents
Kit for extracting blood genome DNA by magnetic bead method and application thereof Download PDFInfo
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- 238000005406 washing Methods 0.000 claims abstract description 52
- -1 tetradecyl sulfobetaine Chemical compound 0.000 claims abstract description 24
- 229940117986 sulfobetaine Drugs 0.000 claims abstract description 23
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 49
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- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims description 27
- 239000003480 eluent Substances 0.000 claims description 23
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 22
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- IZWSFJTYBVKZNK-UHFFFAOYSA-N lauryl sulfobetaine Chemical compound CCCCCCCCCCCC[N+](C)(C)CCCS([O-])(=O)=O IZWSFJTYBVKZNK-UHFFFAOYSA-N 0.000 description 2
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Abstract
The invention discloses a kit for extracting blood genome DNA by a magnetic bead method and application thereof. The invention uses sodium sarcosinate and CHAPS to carry out cell lysis by matching with other components, and has low cost and higher efficiency. In addition, tetradecyl sulfobetaine is used as a surfactant to be matched with other components for washing, so that the content of interfering substances is reduced better, and the purity of nucleic acid is improved. The kit can fully extract DNA in a blood sample and remove impurities, and effectively improves the yield and purity of the DNA. The extraction kit is matched with a full-automatic nucleic acid extractor by a magnetic bead method, is simple, convenient and safe to operate, and can meet the extraction requirements of a large number of samples.
Description
Technical Field
The invention relates to the technical field of nucleic acid extraction, in particular to a kit for extracting blood genome DNA by a magnetic bead method and application thereof.
Background
Blood is a common clinical test sample, and extraction of genomic DNA from blood samples is the basis for clinical tests and biomedical research. However, the components of blood samples are complex, including plasma, erythrocytes, leukocytes and platelets, with most of the genomic DNA present in leukocytes, whereas the volume fraction of leukocytes containing genomic DNA is only around 1%, with a low nucleic acid content. Therefore, it is important to obtain a stable high concentration and high purity genomic DNA from a blood sample rapidly.
Traditional nucleic acid extraction methods, such as phenol-chloroform extraction, boiling cleavage, CTAB, chromatography, etc., generally have the disadvantages of complex operation steps, long time consumption, incapability of automatic operation, low extraction efficiency, and the like, and some of the methods even require the use of toxic organic reagents, which have great influence on the environment and the health of operators. Compared with the traditional nucleic acid purification method, the magnetic bead method nucleic acid extraction technology is attracting attention as a simple, rapid, reliable and automatable nucleic acid extraction method.
The Chinese patent No. 116769770A discloses a kit and a method for extracting genomic DNA of blood tissue cells, wherein under the combined action of a lysate, a washing solution, an eluent, a magnetic bead suspension and a proteinase K solution, the total amount of the extracted DNA is about 10 mug, the A260/A280 is about 1.80, the A260/A230 is above 2.0, and the concentration and the purity are better. However, this experimental method requires manual operation, is not suitable for high throughput processing of large-scale samples, and contains proteinase K in the kit, which requires low-temperature transportation, thus not only increasing the cost, but also leading to enzyme inactivation if improperly preserved, and finally greatly compromising the extraction effect.
Chinese patent No. 116334073A discloses a blood alcohol-free extraction kit based on a magnetic bead method and a use method, wherein the method can be adapted to a full-automatic nucleic acid extractor, and a rinsing liquid adopts an alcohol-free formula, so that the safety performance of an experiment is improved. However, the kit also uses proteinase K, and the extracted nucleic acid A260/A230 is about 1.70 and does not reach the optimal range of 2.0-2.3, one of A260/A280 even exceeds 2.0, and RNA pollution exists, so that the purity of DNA extracted by the kit still needs to be further improved.
Disclosure of Invention
The invention aims to solve the technical problems and defects in the prior art and provides a kit for extracting blood genome DNA by a magnetic bead method and application thereof.
The first object of the present invention is to provide a lysate for extracting blood genomic DNA by a magnetic bead method.
The second object of the invention is to provide the application of the lysate in preparing a kit for extracting blood genome DNA by a magnetic bead method.
The third object of the invention is to provide a kit for extracting blood genome DNA by a magnetic bead method.
In order to achieve the above object, the present invention is realized by the following means:
the invention provides a kit for extracting blood genome DNA by a magnetic bead method. The kit consists of a lysate, magnetic beads, a washing solution I, a washing solution II and an eluent, wherein the lysate comprises the following components: guanidine hydrochloride, PEG8000, sodium iodide, tris-HCl, EDTA, CHAPS, sodium sarcosinate; the magnetic beads are silicon hydroxyl magnetic beads; the washing liquid I comprises the following components: guanidine hydrochloride, sodium chloride, tris-HCl, EDTA, tetradecyl sulfobetaine, triton X-100, isopropanol; the washing liquid II comprises the following components: ethanol, tris-HCl, EDTA; the eluent comprises the following components: tris-HCl, EDTA.
In the kit, the protein is destroyed by the lysate without adding proteinase K, but the protein with high efficiency and nucleic acid connection is destroyed by the synergistic effect of the protein strong denaturant (guanidine hydrochloride) and the surfactant (sodium sarcosinate, CHAPS) so that the nucleic acid is fully released. Simultaneously, sodium iodide can also denature protein, so that the protein is denatured and precipitated, and the nucleic acid can be free from protein entanglement. After the blood sample is cracked, the enzyme inhibitor EDTA can be complexed with metal ions, so that the influence of the metal ions on nucleic acid is avoided; the Tris-HCl buffer solution can provide a more proper cracking environment, the PEG8000 is favorable for the precipitation and enrichment of nucleic acid in the cracking solution, and the PEG8000 is matched with magnetic beads, so that the selectivity of adsorbing nucleic acid is improved, the impurities adsorbed by the magnetic beads are reduced, and the extraction rate and purity of the nucleic acid are improved.
The cleaning solution I is added with a zwitterionic surfactant (tetradecyl sulfobetaine) which has excellent cleaning property, stability, alkali resistance and electrolyte resistance; triton X-100 is a nonionic surfactant and mainly used for dissolving lipid on the surface of a sample so as to enable the sample to be cracked more rapidly and fully; the effect of isopropanol is mainly to precipitate or precipitate DNA. The substances such as guanidine hydrochloride, triton X-100, tetradecyl sulfobetaine, isopropanol and the like are matched for use, so that the content of interfering substances such as protein, guanidine salt and the like can be better reduced, and the purity of nucleic acid can be improved.
In the washing solution II, the ethanol is not cleaned if the ethanol is added too little, so that impurities are generated, and if the ethanol is added too much, the ethanol is not volatilized thoroughly, so that a certain inhibition effect is generated on the subsequent amplification. The 70% ethanol solution, tris-HCl and EDTA can be used together to clean guanidine salt and other impurities and maintain the stability of nucleic acid.
Accordingly, the present invention claims the following:
a lysate for extracting blood genomic DNA by a magnetic bead method, the lysate comprising the following components: 3 to 7 percent M guanidine hydrochloride, 4 to 10 percent PEG8000 (w/v), 0.3 to 0.7 percent M sodium iodide, 50 to 100 mM Tris-HCl, 50 to 80 mM EDTA, 5 to 10 percent CHAPS (w/v), 0.5 to 2 percent sodium sarcosinate (w/v) and pH 7.0 to 9.0.
Preferably, the lysate contains the following components: 4-6-M guanidine hydrochloride, 5-8% PEG8000 (w/v), 0.4-0.6-M sodium iodide, 70-90 mM Tris-HCl, 50-70 mM EDTA, 5-10% CHAPS (w/v), 0.5-2% sodium sarcosinate (w/v), pH 7.5-8.5.
More preferably, the lysate contains the following components: 6M guanidine hydrochloride, 8% PEG8000 (w/v), 0.6M sodium iodide, 80 mM Tris-HCl, 60 mM EDTA, 10% CHAPS (w/v), 2% sodium sarcosinate (w/v), pH 8.2.
The application of any of the above lysate in preparing a kit for extracting blood genomic DNA by a magnetic bead method.
A kit for extracting blood genome DNA by a magnetic bead method, wherein the kit contains any one of the lysate.
Preferably, the kit further comprises magnetic beads.
More preferably, the magnetic beads are silica hydroxyl magnetic beads.
Preferably, the kit also contains a washing liquid I;
the washing liquid I contains the following components: 1 to 3. 3M guanidine hydrochloride, 0.5 to 1.5M sodium chloride, 30 to 50 mM Tris-HCl, 10 to 30 mM EDTA, 5 to 7% tetradecyl sulfobetaine (w/v), 1 to 3% Triton X-100 (w/v), 15 to 25% isopropyl alcohol solution (v/v), and pH 7.5 to 8.0.
More preferably, the washing liquid i contains the following components: 2M guanidine hydrochloride, 1M sodium chloride, 40 mM Tris-HCl, 20 mM EDTA, 6% tetradecyl sulfobetaine (w/v), 2% Triton X-100 (w/v), 20% isopropyl alcohol solution (v/v), pH 7.8.
Preferably, the kit also contains a washing liquid II;
the washing liquid II comprises the following components: 70-75% ethanol solution (v/v), 55-65 mM Tris-HCl, 5-15 mM EDTA.
More preferably, the washing liquid II contains the following components: 70% ethanol (v/v), 60 mM Tris-HCl, 10 mM EDTA.
Preferably, the kit further comprises an eluent;
the eluent contains the following components: 5-15 mM Tris-HCl, 0.5-1.5 mM EDTA,pH 7.5-8.5.
More preferably, the eluent contains the following components: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses a kit for extracting blood genome DNA by a magnetic bead method and application thereof. The kit consists of a lysate, magnetic beads, a washing solution I, a washing solution II and an eluent, wherein the lysate comprises the following components: guanidine hydrochloride, PEG8000, sodium iodide, tris-HCl, EDTA, CHAPS, sodium sarcosinate; the magnetic beads are silicon hydroxyl magnetic beads; the washing liquid I comprises the following components: guanidine hydrochloride, sodium chloride, tris-HCl, EDTA, tetradecyl sulfobetaine, triton X-100, isopropanol; the washing liquid II comprises the following components: ethanol, tris-HCl, EDTA; the eluent comprises the following components: tris-HCl, EDTA.
Compared with the existing products, the kit provided by the invention has at least the following advantages:
(1) The kit does not use toxic reagents such as chloroform, phenol and the like, adopts a pre-packaged reagent plate, is adaptive to a full-automatic nucleic acid extractor, can meet the high-throughput treatment of a large number of samples, and reduces personnel operation steps, thereby greatly reducing the harm to the environment and operators.
(2) The sodium sarcosinate and CHAPS are matched with other components of the invention in the lysate, so that the cell membrane structure can be damaged more efficiently, the nucleic acid can be released more efficiently and rapidly, the lysis efficiency is increased, the extraction amount of the nucleic acid is increased, the A260/A230 value of the nucleic acid is increased, and the nucleic acid is kept in a good purity range. In addition, proteinase K is not needed to be additionally used, so that the experimental cost is effectively reduced, and the experimental operation efficiency is improved.
(3) The tetradecyl sulfobetaine is a zwitterionic surfactant, has excellent cleaning performance, and can better reduce the content of interfering substances and improve the purity of nucleic acid by matching with other components of the invention in the cleaning solution.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The magnetic beads used in the examples are GNT-201 silicon hydroxyl magnetic beads of Roche Jien biotechnology Co., ltd, and the apparatus used for extracting nucleic acid is Kaplan full-automatic nucleic acid extractor HBNP-4801A.
Example 1A kit for extracting genomic DNA from blood by magnetic bead method
1. Composition of components
The kit comprises a lysate, magnetic beads, a washing solution I, a washing solution II and an eluent.
The lysate comprises the following components: 6M guanidine hydrochloride, 8% PEG8000 (w/v), 0.6M sodium iodide, 80 mM Tris-HCl, 60 mM EDTA, 10% CHAPS (w/v), 2% sodium sarcosinate (w/v), pH 8.2.
The concentration of GNT-201 silica-hydroxyl magnetic beads was 2 mg/mL.
The washing liquid I comprises the following components: 2M guanidine hydrochloride, 1M sodium chloride, 40 mM Tris-HCl, 20 mM EDTA, 6% tetradecyl sulfobetaine (w/v), 2% Triton X-100 (w/v), 20% isopropyl alcohol solution (v/v), pH 7.8.
The washing liquid II comprises the following components: 70% ethanol (v/v), 60 mM Tris-HCl, 10 mM EDTA.
The eluent comprises the following components: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.
2. Application method
1. And (3) sub-packaging each component in the kit into a pre-packaging reagent plate of a 96-well plate, wherein the 1 st column and the 7 th column of the pre-packaging reagent plate are internally provided with a lysate, the 2 nd column and the 8 th column of the deep-hole plates are internally provided with a washing liquid I, the 3 rd column and the 9 th column of the deep-hole plates are internally provided with a washing liquid II, the 4 th column and the 11 th column of the deep-hole plates are internally provided with magnetic bead solutions, and the 6 th column and the 12 th column of the deep-hole plates are internally provided with eluent, and the elution is specifically shown in table 1.
Table 1 pre-packed reagent plate
2. 300. Mu.L of blood sample was added to each of columns 1 and 7.
3. Opening a Kaipu full-automatic nucleic acid extractor HBNP-4801A instrument, putting a pre-packed reagent plate added with a sample into the extractor, and then inserting a magnetic rod sleeve.
4. The corresponding nucleic acid extraction program was selected and the extraction program was run, as shown in tables 2 and 3, with both extraction programs running simultaneously.
Table 2 extraction procedure 1
TABLE 3 extraction procedure 2
5. After the automatic procedure is finished, the pre-packaged reagent plate and the magnetic sleeve are taken out, and the nucleic acid solution obtained by extraction in the 6 th column and the 12 th column is taken out for further detection or stored at-20+/-5 ℃.
6. A2 mu L nucleic acid solution is adopted to carry out fluorescence spectrophotometry detection by using Nanodrop 2000 to obtain the ratio of A260/A280 and A260/A230, the ratio of A260/A280 is less than or equal to 1.8 and less than or equal to 2.0 of a pure DNA sample, if the ratio is less than 1.8, the influence of protein or phenolic substances exists, the ratio of A260/A230 is more than 2.0, and if the ratio is less than 2.0, the existence of pollutants such as carbohydrate, guanidine salt and the like in the sample is indicated.
Example 2A kit for extracting genomic DNA from blood by magnetic bead method
1. Composition of components
The kit comprises a lysate, magnetic beads, a washing solution I, a washing solution II and an eluent.
The lysate comprises the following components: 6M guanidine hydrochloride, 8% PEG8000 (w/v), 0.6M sodium iodide, 80 mM Tris-HCl, 60 mM EDTA, 10% CHAPS (w/v), 0.5% sodium sarcosinate (w/v), pH 8.2.
The concentration of GNT-201 silica-hydroxyl magnetic beads was 2 mg/mL.
The washing liquid I comprises the following components: 2M guanidine hydrochloride, 1M sodium chloride, 40 mM Tris-HCl, 20 mM EDTA, 6% tetradecyl sulfobetaine (w/v), 2% Triton X-100 (w/v), 20% isopropyl alcohol solution (v/v), pH 7.8.
The washing liquid II comprises the following components: 70% ethanol (v/v), 60 mM Tris-HCl, 10 mM EDTA.
The eluent comprises the following components: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.
2. Application method
The use was made according to the method of use of example 1.
Example 3A kit for extracting genomic DNA from blood by magnetic bead method
1. Composition of components
The kit comprises a lysate, magnetic beads, a washing solution I, a washing solution II and an eluent.
The lysate comprises the following components: 6M guanidine hydrochloride, 8% PEG8000 (w/v), 0.6M sodium iodide, 80 mM Tris-HCl, 60 mM EDTA, 5% CHAPS (w/v), 2% sodium sarcosinate (w/v), pH 8.2.
The concentration of GNT-201 silica-hydroxyl magnetic beads was 2 mg/mL.
The washing liquid I comprises the following components: 2M guanidine hydrochloride, 1M sodium chloride, 40 mM Tris-HCl, 20 mM EDTA, 6% tetradecyl sulfobetaine (w/v), 2% Triton X-100 (w/v), 20% isopropyl alcohol solution (v/v), pH 7.8.
The washing liquid II comprises the following components: 70% ethanol (v/v), 60 mM Tris-HCl, 10 mM EDTA.
The eluent comprises the following components: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.
2. Application method
The use was made according to the method of use of example 1.
Example 4A kit for extracting genomic DNA from blood by magnetic bead method
1. Composition of components
The kit comprises a lysate, magnetic beads, a washing solution I, a washing solution II and an eluent.
The lysate comprises the following components: 6M guanidine hydrochloride, 8% PEG8000 (w/v), 0.6M sodium iodide, 80 mM Tris-HCl, 60 mM EDTA, 10% CHAPS (w/v), 2% sodium sarcosinate (w/v), pH 8.2.
The concentration of GNT-201 silica-hydroxyl magnetic beads was 2 mg/mL.
The washing liquid I comprises the following components: 2M guanidine hydrochloride, 1M sodium chloride, 40 mM Tris-HCl, 20 mM EDTA, 3% tetradecyl sulfobetaine (w/v), 2% Triton X-100 (w/v), 20% isopropyl alcohol solution (v/v), pH 7.8.
The washing liquid II comprises the following components: 70% ethanol (v/v), 60 mM Tris-HCl, 10 mM EDTA.
The eluent comprises the following components: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.
2. Application method
The use was made according to the method of use of example 1.
Example 5A kit for extracting genomic DNA from blood by magnetic bead method
1. Composition of components
The kit comprises a lysate, magnetic beads, a washing solution I, a washing solution II and an eluent.
The lysate comprises the following components: 5M guanidine hydrochloride, 8% PEG8000 (w/v), 0.6M sodium iodide, 80 mM Tris-HCl, 60 mM EDTA, 10% CHAPS (w/v), 1.5% sodium sarcosinate (w/v), pH 8.2.
The concentration of GNT-201 silica-hydroxyl magnetic beads was 2 mg/mL.
The washing liquid I comprises the following components: 2M guanidine hydrochloride, 1M sodium chloride, 40 mM Tris-HCl, 20 mM EDTA, 6% tetradecyl sulfobetaine (w/v), 2% Triton X-100 (w/v), 20% isopropyl alcohol solution (v/v), pH 7.8.
The washing liquid II comprises the following components: 70% ethanol (v/v), 60 mM Tris-HCl, 10 mM EDTA.
The eluent comprises the following components: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.
2. Application method
The use was made according to the method of use of example 1.
Example 6A kit for extracting genomic DNA from blood by magnetic bead method
1. Composition of components
The kit comprises a lysate, magnetic beads, a washing solution I, a washing solution II and an eluent.
The lysate comprises the following components: 4M guanidine hydrochloride, 8% PEG8000 (w/v), 0.6M sodium iodide, 80 mM Tris-HCl, 60 mM EDTA, 10% CHAPS (w/v), 1% sodium sarcosinate (w/v), pH 8.2.
The concentration of GNT-201 silica-hydroxyl magnetic beads was 2 mg/mL.
The washing liquid I comprises the following components: 2M guanidine hydrochloride, 1M sodium chloride, 40 mM Tris-HCl, 20 mM EDTA, 6% tetradecyl sulfobetaine (w/v), 2% Triton X-100 (w/v), 20% isopropyl alcohol solution (v/v), pH 7.8.
The washing liquid II comprises the following components: 70% ethanol (v/v), 60 mM Tris-HCl, 10 mM EDTA.
The eluent comprises the following components: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.
2. Application method
The use was made according to the method of use of example 1.
Comparative example 1
The difference between this comparative example and example 1 is that the lysate does not contain sodium sarcosinate, otherwise the same as in example 1.
Comparative example 2
This comparative example differs from example 1 in that the lysate does not contain CHAPS, otherwise the same as in example 1.
Comparative example 3
The difference between this comparative example and example 1 is that the lysate does not contain sodium sarcosinate and CHAPS, otherwise the same as in example 1.
Comparative example 4
The difference between this comparative example and example 1 is that PEG8000 in the lysate was replaced with PEG3000 in the same mass to volume ratio as in example 1.
Comparative example 5
The difference between this comparative example and example 1 is that PEG8000 in the lysate was replaced with PEG6000 in the same mass/volume ratio as in example 1.
Comparative example 6
The difference between this comparative example and example 1 is that PEG8000 in the lysate is replaced with PEG10000 in equal mass/volume ratio, otherwise the same as in example 1.
Comparative example 7
The difference between this comparative example and example 1 is that PEG8000 in the lysate was replaced with PEG12000 in the same mass/volume ratio as in example 1.
Comparative example 8
The difference between this comparative example and example 1 is that the washing solution I does not contain tetradecyl sulfobetaine, and the other is the same as in example 1.
Comparative example 9
The difference between this comparative example and example 1 is that the tetradecyl sulfobetaine in the washing solution I was changed to NP40 in an equal mass to volume ratio, and the same as in example 1 was repeated.
Comparative example 10
The difference between this comparative example and example 1 is that the tetradecyl sulfobetaine in the washing solution I was changed to Tween 20 in an equal mass to volume ratio, and the same as in example 1.
Comparative example 11
The difference between this comparative example and example 1 is that the tetradecyl sulfobetaine in the washing solution I was changed to dodecyl sulfobetaine in the same mass-to-volume ratio, and the other is the same as in example 1.
Comparative example 12
The difference between this comparative example and example 1 is that the lysate does not contain sodium sarcosinate and CHAPS, the wash solution I does not contain tetradecyl sulfobetaine, and the other is the same as in example 1.
Performance testing
Nucleic acid extraction was performed using the kits of examples 1 to 6 and comparative examples 1 to 12, and the concentration and purity of the extracted nucleic acid are shown in Table 4.
TABLE 4 nucleic acid concentration and purity
The data in Table 4 shows that the blood genomic DNA extracted by the kit provided in examples 1 to 6, especially example 1, has high concentration and high purity, and the components in example 1 fully exert synergistic effect, and the DNA with high concentration, little influence of protein or phenolic substances and little pollution of guanidine salt is extracted by the extraction method.
The data of example 1 and comparative examples 1-3 show that after sodium sarcosinate and CHAPS are added into the lysate, they fully exert synergistic effect to enhance the lysis, and not only the concentration of extracted nucleic acid is increased, but also the ratio of A260/A280 and A260/A230 is increased.
The results of the data in example 1 and comparative examples 4 to 7 show that the addition of PEG8000 to the lysate is better than the addition of PEG63000, PEG6000, PEG10000, and PEG12000, and the extracted DNA has higher concentration and better purity.
The data of example 1, comparative example 8 and comparative example 12 show that the lysate is sufficiently lysed and the wash solution is sufficiently washed, resulting in a high yield and good purity of nucleic acids. Comparative example 8 in which sodium sarcosinate, CHAPS and tetradecyl sulfobetaine were not added, the washing effect was impaired, and residues of carbohydrates, guanidine salts and the like were also present, resulting in a decrease in a260/a 230. In addition, the tetradecyl sulfobetaine is replaced by other surfactants such as NP40, tween 20, dodecyl sulfobetaine and the like, the cleaning effect is not as good as that of the tetradecyl sulfobetaine, and the A260/A230 does not reach more than 2.0.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (5)
1. A kit for extracting blood genome DNA by a magnetic bead method, which is characterized in that the kit contains a lysate;
the lysate contains the following components: 3 to 7 percent M guanidine hydrochloride, 4 to 10 percent PEG8000 (w/v), 0.3 to 0.7 percent M sodium iodide, 50 to 100 mM Tris-HCl, 50 to 80 mM EDTA, 5 to 10 percent CHAPS (w/v), 0.5 to 2 percent sodium sarcosinate (w/v) and pH 7.0 to 9.0;
the kit also contains a washing liquid I;
the washing liquid I contains the following components: 1 to 3. 3M guanidine hydrochloride, 0.5 to 1.5M sodium chloride, 30 to 50 mM Tris-HCl, 10 to 30 mM EDTA, 5 to 7% tetradecyl sulfobetaine (w/v), 1 to 3% Triton X-100 (w/v), 15 to 25% isopropyl alcohol solution (v/v), pH 7.5 to 8.0;
the kit also contains a washing liquid II;
the washing liquid II comprises the following components: 70-75% ethanol solution (v/v), 55-65 mM Tris-HCl, 5-15 mM EDTA;
the kit also contains eluent;
the eluent contains the following components: 5-15 mM Tris-HCl, 0.5-1.5 mM EDTA,pH 7.5-8.5.
2. The kit of claim 1, wherein the lysate comprises the following components: 4-6-M guanidine hydrochloride, 5-8% PEG8000 (w/v), 0.4-0.6-M sodium iodide, 70-90 mM Tris-HCl, 50-70 mM EDTA, 5-10% CHAPS (w/v), 0.5-2% sodium sarcosinate (w/v), pH 7.5-8.5.
3. The kit of claim 1, wherein the lysate comprises the following components: 6M guanidine hydrochloride, 8% PEG8000 (w/v), 0.6M sodium iodide, 80 mM Tris-HCl, 60 mM EDTA, 10% CHAPS (w/v), 2% sodium sarcosinate (w/v), pH 8.2.
4. A kit according to any one of claims 1 to 3, further comprising magnetic beads.
5. The kit of claim 4, wherein the magnetic beads are silica hydroxyl magnetic beads.
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