CN113265397A - Cell lysate, kit and method for yeast genome extraction - Google Patents
Cell lysate, kit and method for yeast genome extraction Download PDFInfo
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- CN113265397A CN113265397A CN202110609354.6A CN202110609354A CN113265397A CN 113265397 A CN113265397 A CN 113265397A CN 202110609354 A CN202110609354 A CN 202110609354A CN 113265397 A CN113265397 A CN 113265397A
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- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 41
- 239000013592 cell lysate Substances 0.000 title claims abstract description 25
- 238000000605 extraction Methods 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 23
- 239000007983 Tris buffer Substances 0.000 claims abstract description 9
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 claims abstract description 9
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000000243 solution Substances 0.000 claims description 38
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 23
- 238000001179 sorption measurement Methods 0.000 claims description 19
- 239000007788 liquid Substances 0.000 claims description 16
- 238000002156 mixing Methods 0.000 claims description 13
- 239000007853 buffer solution Substances 0.000 claims description 12
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 10
- 108010067770 Endopeptidase K Proteins 0.000 claims description 10
- 239000000600 sorbitol Substances 0.000 claims description 10
- 239000000872 buffer Substances 0.000 claims description 9
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 claims description 7
- 102000005891 Pancreatic ribonuclease Human genes 0.000 claims description 7
- 235000019441 ethanol Nutrition 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 239000002699 waste material Substances 0.000 claims description 6
- 241001052560 Thallis Species 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 239000007984 Tris EDTA buffer Substances 0.000 claims description 3
- 229920000136 polysorbate Polymers 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 2
- 239000000725 suspension Substances 0.000 claims 1
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 abstract description 6
- 238000007400 DNA extraction Methods 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract description 3
- 108020004414 DNA Proteins 0.000 description 20
- 210000004027 cell Anatomy 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 229920000057 Mannan Polymers 0.000 description 3
- 230000006037 cell lysis Effects 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 210000005253 yeast cell Anatomy 0.000 description 3
- 229920001503 Glucan Polymers 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000033383 cell-cell recognition Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002052 molecular layer Substances 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
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Abstract
The invention relates to the technical field of DNA extraction, and provides a cell lysate, a kit and a method for yeast genome extraction. The cell lysate for yeast genome extraction comprises the following components in percentage by weight: 50-60 g/L of guanidinium isothiocyanate, 1-10 g/L of Tris-base, 8-15 g/L of EDTA-2 Na and 201-10% of Tween-L. Compared with the prior art, the yeast genome extraction kit provided by the invention is time-saving and labor-saving, the cells are thoroughly cracked, chloroform is avoided in the experimental process, the harm to experimental operators is reduced, the extracted genome yield is high, the kit can be directly used for subsequent experiments, and the genome does not need to be purified.
Description
Technical Field
The invention relates to the technical field of DNA extraction, in particular to a cell lysate for yeast genome extraction, a kit containing the lysate and a method for extracting a yeast genome by using the kit.
Background
Yeast is often used as a model system for eukaryotic genome expression due to its characteristics of clear genetic background, simple operation, rapid growth and the like. In addition, yeast is also widely used in genetic engineering and food production. In the process of researching yeast, extracting the genome of the yeast to detect the target gene is an important step.
The yeast cell wall is tough and thick, the thickness is 0.1-0.3 mu m, the weight accounts for 18-30% of the dry weight of the cell, and the yeast cell wall has the important function of maintaining the cell morphology and the cell-cell recognition. Its structure is similar to a sandwich. The outer layer is mannan, the middle layer is a protein molecular layer, and the inner layer is glucan. The mannan backbone in the outer layer is combined by alpha-1, 6 glycosidic bond, the side chain is combined by alpha-1, 2 or alpha-1, 3 glycosidic bond, the glucan in the inner layer is combined by beta-1, 3 glycosidic bond to form a net structure, the strength and toughness of cell wall are maintained, and the mannan can be greatly extended under normal osmotic pressure. Efficient disruption of the yeast cell wall is therefore critical for successful extraction of the yeast genome.
At present, the extraction of yeast genome is costly, time-consuming and poor in quality, so an improved and optimized yeast genome extraction method and related products are needed.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a cell lysate, a kit and a method for extracting a yeast genome.
In order to achieve the purpose, the invention adopts the following technical scheme:
the first aspect of the invention provides a cell lysate for yeast genome extraction, which comprises the following components in percentage by weight: 50-60 g/L of guanidinium isothiocyanate, 1-10 g/L of Tris-base, 8-15 g/L of EDTA-2 Na and 201-10% of Tween-L.
Further, the cell lysate comprises the following components in percentage by weight: 54.2g/L of guanidinium isothiocyanate, 3.6g/L of Tris-base, 9.4g/L of EDTA-2 Na and 202 percent of Tween.
The second aspect of the present invention provides a kit for yeast genome extraction comprising the above cell lysate.
Further, the kit also comprises sorbitol buffer solution, SDS buffer solution, lywallzyme solution, RNase A solution, proteinase K solution and 7U adsorption columns.
Further, the concentration of sorbitol buffer was 0.1 g/ml.
Further, the concentration of SDS buffer was 0.8%.
Further, the concentration of the lywallzyme solution was 10 mg/ml.
Further, the concentration of the RNase A solution was 15 mg/ml.
Further, the concentration of the proteinase K solution was 20 mg/ml.
The third aspect of the present invention is to provide a method for yeast genome extraction using the above-mentioned kit, comprising the steps of:
taking yeast liquid cultured overnight, centrifuging, and collecting thalli; sequentially adding sorbitol buffer solution and lywallzyme solution into the collected thalli, fully and uniformly mixing and incubating for 20-40 minutes;
centrifuging and discarding the supernatant; adding SDS buffer solution into the precipitate to resuspend the precipitate, then adding RNase A solution, fully and uniformly mixing, and standing for 3-10 minutes at room temperature;
adding cell lysate, mixing uniformly, adding protease K solution, mixing uniformly, and preserving heat at the temperature of 60-80 ℃;
cooling to room temperature, adding absolute ethyl alcohol, fully and uniformly mixing, and adding onto a 7U adsorption column;
step five, centrifuging, discarding the waste liquid in the collecting pipe, adding 80% ethanol solution, centrifuging, taking down the adsorption column, and discarding the waste liquid in the collecting pipe;
step six, repeating the step five; centrifuging to remove residual ethanol; putting the adsorption column into a new clean centrifugal tube, adding 50-100 mu l of TE buffer solution into the center of the adsorption column, and standing at room temperature or 37 ℃;
and seventhly, centrifuging, wherein the liquid in the centrifugal tube is the yeast genome.
Further, the density of yeast liquid is not more than 2.5 × 107Cells/ml; the volume ratio of the yeast liquid to the sorbitol buffer solution to the lywallzyme solution to the SDS buffer solution to the cell lysate to the proteinase K solution is 1: 0.2-0.8: 0.0025-0.005: 0.05-0.3: 0.1-0.2: 0.01-0.02.
Further, the volume ratio of the RNase A solution, the absolute ethyl alcohol and the cell lysate used in the above method is 0.01-0.02: 1: 1.
further, the incubation in step one was carried out at 30 ℃ for 30 minutes.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
(1) the cell lysis solution contains guanidine isothiocyanate and tween-20, and the SDS buffer solution contains SDS, which is beneficial to cell lysis, shortens the time of lysis for extraction and has better DNA extraction effect;
(2) the method provided by the invention simplifies the operation steps, the supernatant generated in the step two can be directly poured out, the supernatant is prevented from being sucked by a liquid-transferring gun, the method is simple and quick, the working hours are saved, and the gun head cost is saved;
(3) chloroform with high toxic odor is avoided, and the harm to the health of operators is reduced;
(4) the extract has high DNA yield and high DNA content, and is especially combined with the use of a 7U adsorption column, so that the protein and salt in the DNA are completely removed, and the quality of the extracted DNA is improved;
(5) the result is quite stable, the success rate is high, and the method is suitable for extracting DNA on a large scale.
In conclusion, compared with the prior art, the yeast genome extraction kit provided by the invention is time-saving and labor-saving, the cell lysis is thorough, the use of chloroform is avoided in the experimental process, and the harm to experimental operators is reduced. The extracted genome has high yield, can be directly used for subsequent experiments, and does not need to purify the genome.
Drawings
FIG. 1 shows the results of agarose gel electrophoresis of the genomes obtained by the method provided in example 5 and the method provided in comparative example 1; wherein, the DNA extracted in comparative example 1 is contained in 1 to 6 wells, and the DNA extracted in example 5 is contained in 7 to 12 wells; m is DNA Marker DL2502 from Shanghai Czeri bioengineering, Inc.
Detailed Description
The invention provides a cell lysate, a kit and a method for yeast genome extraction. The cell lysate comprises the following components in percentage by weight: 50-60 g/L of guanidinium isothiocyanate, 1-10 g/L of Tris-base, 8-15 g/L of EDTA-2 Na and 201-10% of Tween-L.
The present invention will be described in detail and specifically with reference to the following examples and drawings so as to provide a better understanding of the invention, but the following examples do not limit the scope of the invention.
In the examples, the conventional methods were used unless otherwise specified, and reagents used were those conventionally commercially available or formulated according to the conventional methods without specifically specified.
Example 1
The embodiment provides a cell lysate for yeast genome extraction, which comprises the following components in percentage by weight: 54.2g/L, Tris-base 3.6g/L, EDTA.2 Na 9.4g/L guanidinium isothiocyanate, tween-202%.
Example 2
The embodiment provides a cell lysate for yeast genome extraction, which comprises the following components in percentage by weight: 60g/L, Tris-base 2.4g/L, EDTA.2 Na 12g/L guanidinium isothiocyanate, and 205% Tween.
Example 3
The embodiment provides a cell lysate for yeast genome extraction, which comprises the following components in percentage by weight: 50g/L, Tris-base 4.2g/L, EDTA.2 Na 5.4g/L guanidinium isothiocyanate, and tween-201 percent.
Example 4
This example provides a kit for yeast genome extraction comprising the cell lysate of example 1, further comprising reagents and equipment at the following concentrations: sorbitol buffer 0.1g/ml, SDS buffer 0.8%, lywallzyme solution 10mg/ml, RNase A solution 15mg/ml, proteinase K solution 20mg/ml and 7U adsorption column.
Example 5
This example provides a method for yeast genome extraction using the kit provided in example 4, comprising the steps of:
1) taking 15 overnight cultured 2ml yeast liquid (no more than 5 × 10 at most)7Cells), the Cells were put into a 2ml centrifuge tube, centrifuged at 8,000rpm at room temperature for 1 minute, and the supernatant was aspirated as much as possible to collect the Cells.
2) Add 600. mu.l sorbitol buffer to the cells, add 5. mu.l lywallzyme solution, mix well, incubate at 30 ℃ for 30min, preferably mix 1 time every 5 minutes.
3) Centrifuging at 8,000rpm for 5min, discarding the supernatant, collecting the precipitate, and removing the residual solution as completely as possible.
4) The precipitate was resuspended in 200. mu.l of SDS buffer, 4. mu.l of RNase A solution was added, mixed well and left at room temperature for 5 minutes.
5) Adding 220 mul cell lysate, mixing, adding 20 mul proteinase K solution, mixing, and keeping temperature at 70 deg.C for 10 min.
6) And (3) when the sample is cooled to be close to room temperature, adding 220 mu l of absolute ethyl alcohol, fully and uniformly mixing, if precipitation exists, not influencing extraction, uniformly beating by using a gun head, and directly adding the mixture to a 7U adsorption column.
7) Centrifugation at 8,000rpm for 1 minute at room temperature; taking down the adsorption column, discarding the waste liquid in the collection tube, adding 500 μ l of 80% ethanol solution, centrifuging at 8,000rpm at room temperature for 1 min; the adsorption column is taken down, and the waste liquid in the collection tube is discarded. Repeat step 7 once.
8) The adsorption column was returned to the collection tube and centrifuged at 12,000rpm at room temperature for 1 minute to remove residual ethanol.
9) The adsorption column is placed into a new clean 1.5ml centrifuge tube, 50-100. mu.l of TE buffer solution is added into the center of the adsorption column, and the adsorption column is placed for 2 minutes at room temperature or 37 ℃.
10) Centrifuge at 12,000rpm for 1 minute at room temperature. The liquid in the centrifuge tube is the genome DNA. Depending on the application, the samples may be stored at 4 ℃ or-20 ℃.
Comparative example 1
For 15 overnight-cultured 2ml yeast fluid samples, other brands of yeast genome extraction kits (Tiangen DP307) were used and extracted according to the kit instructions.
Verification example 1
In this example, DNA quality detection was performed on the genomes obtained by the methods provided in example 5 and comparative example 1, specifically, agarose gel electrophoresis was performed on the genomes (the results are shown in FIG. 1) and an ultraviolet spectrophotometer was used to measure OD values (the results are shown in Table 1).
The principle of judging quality according to the OD value is as follows: because the DNA has the maximum absorption peak at 260nm, the protein has the maximum absorption peak at 280nm, and the salt and the small molecules are concentrated at 230nm, the DNA concentration can be measured by light splitting with the wavelength of 260 nm; when the 260nmOD value of the double-stranded DNA is 1.0, the corresponding DNA concentration is 50 mu g/ml, and the OD260/OD280 of the pure DNA is 1.8, so that the purity of the DNA can be estimated according to the OD260/OD280 value; the OD260/OD280 ratio of the high-quality DNA is 1.70-2.0; if the ratio is higher, RNA is contained, and the ratio is less than 1.80, protein or phenol impurities exist. The ratio OD230/OD260 should be between 0.4 and 0.5, with a higher ratio indicating the presence of residual salt. The DNA purity (OD260/OD280 ratio), the DNA impurity content (OD260/OD230 ratio) and the DNA concentration (50. mu.g/ml. times. OD 260. times. dilution factor/1000) were calculated from the OD value of the sample to be measured.
TABLE 1 results of concentration measurement
According to the results, the concentration and purity of the yeast genome extracted by the method are high, and the yeast genome meets the requirements of subsequent experiments.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Claims (10)
1. A cell lysate for yeast genome extraction is characterized by comprising the following components in percentage by weight: 50-60 g/L of guanidinium isothiocyanate, 1-10 g/L of Tris-base, 8-15 g/L of EDTA-2 Na and 201-10% of Tween-L.
2. The suspension cell lysate according to claim 1, comprising the following components and their contents: 54.2g/L of guanidinium isothiocyanate, 3.6g/L of Tris-base, 9.4g/L of EDTA-2 Na and 202 percent of Tween.
3. A kit for yeast genome extraction comprising the cell lysate of claim 1 or 2.
4. The kit according to claim 3, further comprising sorbitol buffer, SDS buffer, lywallzyme solution, RNaseA solution, proteinase K solution and 7U adsorption column; the concentration of the sorbitol buffer solution is preferably 0.1 g/ml; the concentration of the SDS buffer is preferably 0.8%.
5. The kit according to claim 3, characterized in that the concentration of the lywallzyme solution is 10 mg/ml.
6. The kit according to claim 3, wherein the concentration of the RNase A solution is 15 mg/ml.
7. The kit according to claim 3, wherein the concentration of the proteinase K solution is 20 mg/ml.
8. A method for yeast genome extraction using the kit according to any one of claims 3 to 7, comprising the steps of:
taking yeast liquid cultured overnight, centrifuging, and collecting thalli; sequentially adding the sorbitol buffer solution and the lywallzyme solution into the collected thalli, fully and uniformly mixing, and incubating for 20-40 minutes;
centrifuging and discarding the supernatant; adding the SDS buffer solution into the precipitate to resuspend the precipitate, then adding the RNaseA solution, fully and uniformly mixing, and standing at room temperature for 3-10 minutes;
adding the cell lysate, uniformly mixing, adding the protease K solution, uniformly mixing, and preserving heat at the temperature of 60-80 ℃;
cooling to room temperature, adding absolute ethyl alcohol, fully and uniformly mixing, and adding the mixture to the 7U adsorption column;
step five, centrifuging, discarding the waste liquid in the collecting pipe, adding 80% ethanol solution, centrifuging, taking down the adsorption column, and discarding the waste liquid in the collecting pipe;
step six, repeating the step five; centrifuging to remove residual ethanol; putting the adsorption column into a new clean centrifugal tube, adding 50-100 mu l of TE buffer solution into the center of the adsorption column, and standing at room temperature or 37 ℃;
and seventhly, centrifuging, wherein the liquid in the centrifugal tube is the yeast genome.
9. The method of claim 8, wherein the yeast liquid has a density of not more than 2.5X 107Cells/ml; the volume ratio of the yeast liquid to the sorbitol buffer solution to the lywallzyme solution to the SDS buffer solution to the cell lysate to the proteinase K solution is 1: 0.2-0.8: 0.0025-0.005: 0.05-0.3: 0.1-0.2: 0.01-0.02.
10. The method according to claim 8, wherein the volume ratio of RNaseA solution, absolute ethanol and cell lysate used in the method is 0.01-0.02: 1: 1.
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| CN114958610A (en) * | 2022-06-21 | 2022-08-30 | 浙大宁波理工学院 | Application of phenyllactic acid in preparation of yeast lysate, kit and method |
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Cited By (2)
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| CN114958610A (en) * | 2022-06-21 | 2022-08-30 | 浙大宁波理工学院 | Application of phenyllactic acid in preparation of yeast lysate, kit and method |
| CN114958610B (en) * | 2022-06-21 | 2023-08-18 | 浙大宁波理工学院 | Application of phenyllactic acid in preparation of saccharomycete lysate, kit and method |
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