CN111378649A - Efficient washing liquid - Google Patents
Efficient washing liquid Download PDFInfo
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- CN111378649A CN111378649A CN202010096626.2A CN202010096626A CN111378649A CN 111378649 A CN111378649 A CN 111378649A CN 202010096626 A CN202010096626 A CN 202010096626A CN 111378649 A CN111378649 A CN 111378649A
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- washing liquid
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- 238000005406 washing Methods 0.000 title claims abstract description 23
- 239000007788 liquid Substances 0.000 title claims abstract description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 39
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 12
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims abstract description 10
- 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 abstract description 8
- 239000011780 sodium chloride Substances 0.000 claims abstract description 8
- 239000002904 solvent Substances 0.000 claims abstract description 8
- 150000007523 nucleic acids Chemical class 0.000 abstract description 18
- 102000039446 nucleic acids Human genes 0.000 abstract description 17
- 108020004707 nucleic acids Proteins 0.000 abstract description 17
- 238000000034 method Methods 0.000 abstract description 7
- 239000003960 organic solvent Substances 0.000 abstract description 6
- 238000002360 preparation method Methods 0.000 abstract description 6
- 108090000623 proteins and genes Proteins 0.000 abstract description 6
- 102000004169 proteins and genes Human genes 0.000 abstract description 6
- 239000012535 impurity Substances 0.000 abstract description 4
- 150000003839 salts Chemical class 0.000 abstract description 4
- 150000001720 carbohydrates Chemical class 0.000 abstract description 3
- 239000013522 chelant Substances 0.000 abstract description 2
- 238000005336 cracking Methods 0.000 abstract description 2
- 150000002500 ions Chemical class 0.000 abstract description 2
- 231100000331 toxic Toxicity 0.000 abstract description 2
- 230000002588 toxic effect Effects 0.000 abstract description 2
- 235000019441 ethanol Nutrition 0.000 description 9
- 238000000605 extraction Methods 0.000 description 7
- 238000001179 sorption measurement Methods 0.000 description 7
- 238000003752 polymerase chain reaction Methods 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 108010067770 Endopeptidase K Proteins 0.000 description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 239000008096 xylene Substances 0.000 description 2
- GNFTZDOKVXKIBK-UHFFFAOYSA-N 3-(2-methoxyethoxy)benzohydrazide Chemical compound COCCOC1=CC=CC(C(=O)NN)=C1 GNFTZDOKVXKIBK-UHFFFAOYSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Plant Pathology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Saccharide Compounds (AREA)
- Molecular Biology (AREA)
Abstract
The invention discloses a high-efficiency washing liquid. It comprises 70-80% V/V ethanol, 50-100 mM NaCl, 0.5-1 mM EDTA, 5-15 mM Tris-HCl, 1-1.5 mg/ml BSA, and DEPC water as solvent. In the washing liquid, EDTA can chelate various ions, ethanol can wash away organic solvent, and BSA can remove protein generated in the cracking process of the sample, thereby obtaining nucleic acid with higher purity. Namely, the invention adopts a high-efficiency washing liquid system, can wash impurity salts, proteins, saccharides, organic solvents and the like, and obtains high-quality and high-purity nucleic acid. The washing liquid system has simple preparation method, easy operation and no toxic and harmful components.
Description
The technical field is as follows:
the invention belongs to the field of DNA extraction, and particularly relates to a high-efficiency washing solution.
Background art:
nucleic acid extraction is the basis of various molecular biology detection methods, and efficient and complete extraction of RNA or DNA is the basis of PCR amplification, library construction, sequencing and other works. However, in the PCR molecular diagnosis process, the total copy number and quality purity of the obtained RNA or DNA directly affect the results of the subsequent test experiments, so that obtaining high-purity and high-quality nucleic acid is the most basic prerequisite for the smooth downstream research.
The kit method is the most simple and convenient method with wide clinical application at present, the commercialized nucleic acid extraction kit has various types and different performances, but has different extraction efficiency, the kit mainly comprises lysis solution, proteinase K solution, binding solution, washing solution and eluent, and can finish the nucleic acid extraction work of a large batch of samples within 2 hours, but the commonly used kit at present often has a problem: 1. the washing solution has poor purification effect, the obtained nucleic acid has high concentration but low purity, and is often doped with many impurities, such as salts, proteins, sugars, organic solvents, and the like, and PCR (Polymerase Chain Reaction) is a molecular biology technology for amplifying specific nucleic acid fragments, and has the greatest characteristic of being capable of enriching and doubling a large amount of trace nucleic acid, thereby achieving the purpose of conveniently detecting the trace nucleic acid. The purity requirement of the nucleic acid is extremely high, and PCR molecular diagnosis is often seriously influenced when the nucleic acid is polluted, so that the development of downstream experiments is influenced.
Therefore, an efficient washing solution is urgently needed to better solve the problem of low purity of nucleic acid.
The invention content is as follows:
the invention aims to provide a formula of a high-efficiency washing solution, which can be used for purifying DNA and RNA and washing out impurity salts, proteins, saccharides, organic solvents and the like to obtain nucleic acid with higher purity.
The efficient washing liquid comprises 70-80% V/V ethanol, 50-100 mM NaCl, 0.5-1 mM EDTA, 5-15 mM Tris-HCl and 1-1.5 mg/ml BSA, and the solvent is DEPC water.
Preferably, the reagent comprises 70% V/V ethanol, 50mM NaCl, 0.5mM EDTA, 10mM Tris-HCl and 1mg/ml BSA, and the solvent is DEPC water.
In the washing liquid, EDTA can chelate various ions, ethanol can wash away organic solvent, and BSA can remove protein generated in the cracking process of the sample, thereby obtaining nucleic acid with higher purity. Namely, the invention adopts a high-efficiency washing liquid system, can wash impurity salts, proteins, saccharides, organic solvents and the like, and obtains high-quality and high-purity nucleic acid. The washing liquid system has simple preparation method, easy operation and no toxic and harmful components.
The specific implementation mode is as follows:
the invention is further illustrated by the following examples. It should be understood that these examples are only for the purpose of the present invention and are not intended to limit the scope of the present invention. Unless defined or indicated otherwise, the scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
Example 1:
the high-efficiency washing liquid comprises the following components: 70% v/v ethanol, 50mM NaCl, 0.5mM EDTA, 10mM Tris-HCl, 1mg/ml BSA, solvent DEPC water. The preparation method is to mix the components evenly according to the content.
RNA was isolated and purified from 100 samples, which were paraffin-embedded clinical specimens within 2 years, and compared in parallel with a common kit.
1. Extraction of RNA
The reagent except the washing liquid is the high-efficiency washing liquid, other reagents are common kit components, and parallel experiments are carried out according to common kit instructions.
The common Kit of the invention is an RNAcure FFPE Kit (fixed tissue RNA extraction Kit), and the catalog number is as follows: CW0535S (50preps), storage conditions: room temperature (15-30 ℃);
the extraction method comprises the following steps:
remarking: buffer RW2 is the wash solution in the RNAApure FFPE Kit, which was changed to the high efficiency wash solution of the present invention when tested.
Product content
Procedure for the preparation of the
a. Taking about 1 × 1cm2The sections (total of about 4-5 sections) were placed in a centrifuge tube (self-contained), 1ml of xylene was added, the tube cap was closed, and vortexed for 10 seconds.
b.12,000rpm for 2 minutes, carefully discard the supernatant, take care not to discard the pellet.
c. Adding 1ml of absolute ethyl alcohol, and uniformly mixing by vortex shaking. Centrifuge at 12,000rpm for 2 minutes, discard the supernatant, and take care not to aspirate the pellet.
Note that: ethanol can remove residual xylene from the sample.
d. The tube caps were opened and incubated at room temperature or up to 37 ℃ for 10 minutes until no ethanol remained.
e. Adding 150. mu.l Buffer GTL, and resuspending the precipitate; add 10. mu.l of Proteinase K and vortex and mix well.
f.56 ℃ for 15 minutes until the sample is completely dissolved. Incubate at 80 ℃ for 15 minutes. The solution on the tube wall was collected to the bottom of the tube by brief centrifugation.
Note that: 1) the purpose of this step is to repair nucleic acids denatured by formaldehyde, and too high a temperature or too long an incubation time may cause RNA fragmentation, resulting in RNA fragments. 2) The sample incubated at 56 ℃ can be placed at room temperature until the temperature of the water bath or the dry bath reaches 80 ℃, and then the sample is placed at 80 ℃ for incubation.
g. Add 320. mu.l Buffer GL and mix thoroughly by vortexing.
h. The solution obtained in step g is added in its entirety to a filtration column (Spin columns fm) which has been filled with collection tubes. Centrifuged at 12,000rpm for 1 minute and the filtrate was collected.
i. And h, adding 720 mu l of absolute ethyl alcohol into the filtrate obtained in the step h, and thoroughly mixing the mixture by vortex oscillation.
Note that: after the absolute ethyl alcohol is added, a small amount of precipitate can be separated out, but the subsequent operation is not influenced.
j. And (e) completely adding the solution obtained in the step (i) into an adsorption column (Spin Columns RS) filled with a collecting tube, and transferring for many times if the solution cannot be added at one time. Centrifuge at 12,000rpm for 1 minute, remove waste from the collection tube, and replace the adsorption column back into the collection tube.
k. Mu.l Buffer RW2 (checked for absolute ethanol addition before use) was added to the adsorption column, centrifuged at 12,000rpm for 1 min, the trap was decanted, and the adsorption column was replaced in the trap.
Repeating step k.
m.12,000rpm for 2 minutes and the waste liquid in the collection tube was decanted. The column was left at room temperature for several minutes to dry thoroughly.
Note that: the purpose of this step is to remove residual ethanol from the column, which could affect subsequent enzymatic reactions (enzymatic cleavage, PCR, etc.).
n. placing the adsorption column in a new RNase-Free centrifuge tube, suspending 20-50 μ l RNase-Free Water in the middle part of the adsorption column, placing for 2-5 minutes at room temperature, centrifuging for 1 minute at 12,000rpm, collecting RNA solution, and storing RNA at-20 ℃.
Note that: 1) the RNase-Free Water volume should not be less than 20. mu.l, and too small a volume affects recovery.
2) If the RNA yield is to be increased, step n may be repeated with 20-50. mu.l of a new RNase-Free Water.
3) If the RNA concentration is to be increased, the resulting solution may be re-applied to the adsorption column and step n repeated.
2. Detection of the RNA content
The content and purity of RNA extracted by the high-efficiency washing solution are detected by Nondrop one, and the results are shown in Table 1:
TABLE 1 RNA content and purity extracted by the method of the invention and by the usual kit
The result shows that the purity of the nucleic acid purified by the high-efficiency washing solution is obviously higher than that of the common kit.
Example 2:
this example is essentially the same as example 1, except that the high efficiency wash liquor components include: 80% v/v ethanol, 100mM NaCl, 1mM EDTA, 5mM Tris-HCl, 1mg/ml BSA, solvent DEPC water. The preparation method is to mix the components evenly according to the content.
Example 3:
this example is essentially the same as example 1, except that the high efficiency wash liquor components include: 80% v/v ethanol, 100mM NaCl, 1mM EDTA, 15mM Tris-HCl, 1mg/ml BSA, solvent DEPC water. The preparation method is to mix the components evenly according to the content.
Claims (2)
1. The efficient washing liquid is characterized by comprising 70-80% of V/V ethanol, 50-100 mM NaCl, 0.5-1 mM EDTA, 5-15 mM Tris-HCl and 1-1.5 mg/ml BSA, and the solvent is DEPC water.
2. The high efficiency washing solution according to claim 1, comprising 70% V/V ethanol, 50mM NaCl, 0.5mM EDTA, 10mM Tris-HCl, 1mg/ml BSA, and the solvent is DEPC water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010096626.2A CN111378649A (en) | 2020-02-17 | 2020-02-17 | Efficient washing liquid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010096626.2A CN111378649A (en) | 2020-02-17 | 2020-02-17 | Efficient washing liquid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111378649A true CN111378649A (en) | 2020-07-07 |
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| CN202010096626.2A Pending CN111378649A (en) | 2020-02-17 | 2020-02-17 | Efficient washing liquid |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050042656A1 (en) * | 2003-07-09 | 2005-02-24 | Davis James C. | Room temperature elution of nucleic acids |
| CN102911932A (en) * | 2012-10-25 | 2013-02-06 | 中华人民共和国北京出入境检验检疫局 | Assay kit and method for simultaneously extracting and purifying ribose nucleic acid (RNA) and deoxyribose nucleic acid (DNA) |
| CN106191036A (en) * | 2016-08-08 | 2016-12-07 | 吴江近岸蛋白质科技有限公司 | Rna binding protein is at the application extracted in nucleic acid and extracting method |
| CN108949747A (en) * | 2018-07-27 | 2018-12-07 | 广州奇辉生物科技有限公司 | A kind of kit and application method extracting RNA |
| CN109022417A (en) * | 2018-08-13 | 2018-12-18 | 益善生物技术股份有限公司 | A kind of paramagnetic particle method nucleic acid extraction conversion reagent box and its application method |
| CN110511925A (en) * | 2019-08-29 | 2019-11-29 | 无锡市第二人民医院 | A kind of column method whole blood nucleic acid preservation extracts integrated kit and extracting method |
-
2020
- 2020-02-17 CN CN202010096626.2A patent/CN111378649A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050042656A1 (en) * | 2003-07-09 | 2005-02-24 | Davis James C. | Room temperature elution of nucleic acids |
| CN102911932A (en) * | 2012-10-25 | 2013-02-06 | 中华人民共和国北京出入境检验检疫局 | Assay kit and method for simultaneously extracting and purifying ribose nucleic acid (RNA) and deoxyribose nucleic acid (DNA) |
| CN106191036A (en) * | 2016-08-08 | 2016-12-07 | 吴江近岸蛋白质科技有限公司 | Rna binding protein is at the application extracted in nucleic acid and extracting method |
| CN108949747A (en) * | 2018-07-27 | 2018-12-07 | 广州奇辉生物科技有限公司 | A kind of kit and application method extracting RNA |
| CN109022417A (en) * | 2018-08-13 | 2018-12-18 | 益善生物技术股份有限公司 | A kind of paramagnetic particle method nucleic acid extraction conversion reagent box and its application method |
| CN110511925A (en) * | 2019-08-29 | 2019-11-29 | 无锡市第二人民医院 | A kind of column method whole blood nucleic acid preservation extracts integrated kit and extracting method |
Non-Patent Citations (1)
| Title |
|---|
| 郑育声: "《现代生物技术实验指南：分子生物学与生物化学》", 31 July 2011 * |
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Application publication date: 20200707 |