CN106978414A - A kind of nucleic acid-protein extracts reagent and its application and nucleic acid-protein extracting method - Google Patents
A kind of nucleic acid-protein extracts reagent and its application and nucleic acid-protein extracting method Download PDFInfo
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- CN106978414A CN106978414A CN201710124470.2A CN201710124470A CN106978414A CN 106978414 A CN106978414 A CN 106978414A CN 201710124470 A CN201710124470 A CN 201710124470A CN 106978414 A CN106978414 A CN 106978414A
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- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
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Abstract
The present invention relates to nucleic acid-protein extracts reagent, it includes 0.1 0.8M sodium chloride, 0.3 1.0M ammonium sulfate, the phenol of 0.7 1.5M guanidine hydrochloride and volume fraction 10% 50%；Further relate to application of the above-mentioned nucleic acid-protein extracts reagent in nucleic acid and/or Protein Extraction；Further relate to one kind and nucleic acid and/or method of protein are extracted from histocyte sample.The nucleic acid-protein extracts reagent and method extraction efficiency of the present invention is high, and the integrality for obtaining nucleic acid and protein is good, and extracting method is simple and convenient, is difficult contaminated samples, also will not be to human body occurrence injury.
Description
Technical field
The present invention relates to molecular biology reagents field, more particularly, it is related to a kind of nucleic acid-protein extracts reagent and carries
The method for taking nucleic acid and/or albumen.
Background technology
It is the significant process in genetic engineering or clinical examination field that nucleic acid is extracted from biological sample.For example, from people
When blood or tissue are gathered in body etc. and it being tested, it is necessary to only nucleic acid is extracted from the Biosamples such as blood,
Purifying.For protein isolate matter or nucleic acid from other compositions, after the dissolution process that physics and chemistry are carried out to Biosample, lead to
Extraction operation is crossed to come protein degradation matter or lipid and produce nucleic acid to dissociate.Now, the mistake of protein or nucleic acid is being isolated and purified
Cheng Zhong, easily causes the pollution of sample.As the standard method of extraction generally using at organic solvent, but its toxicity, discarded object
Reason will take time and energy, and centrifuge that operation will also take time and energy and the pollution to sample or the infection from sample turn into and asked
Topic.
The content of the invention
To solve problem above, the invention provides a kind of nucleic acid-protein extracts reagent, it includes 0.1-0.8M chlorination
Sodium, 0.3-1.0M ammonium sulfate, 0.7-1.5M guanidine hydrochloride and volume fraction are 10-50% phenol.
Present invention also offers application of the above-mentioned nucleic acid-protein extracts reagent in nucleic acid and/or Protein Extraction.
Nucleic acid and/or method of protein are extracted from histocyte sample present invention also offers one kind, it include with
Lower step：
S1：By the histocyte sample broke into homogenate；
S2：The organic solvent diluting liquid of the nucleic acid-protein extracts reagent described in claim 1 is added into the homogenate, is mixed
It is even, obtain extracting mixed liquor；
S3：The extraction mixed liquor is centrifuged, layering obtains aqueous phase, middle level and organic phase；
S4：The nucleic acid and/or protein of the histocyte sample are extracted from corresponding layer.
Preferably, the histocyte sample and the ratio of the amount of the nucleic acid-protein extracts reagent are that every 50-100mg is organized
Nucleic acid-protein extracts reagent described in cell sample addition 1ml.
Preferably, the organic solvent is chloroform, and the volume ratio of the nucleic acid-protein reagent and chloroform is 1：0.1-
0.5。
Further, to be extracted for RNA, extracted from the aqueous phase, and S4 is specifically included：
S411：The aqueous phase in S3 is taken, adding isopropanol separates out RNA；
S412：Precipitated after centrifugation, centrifugation is washed with the ethanol of volume fraction 75%, the i.e. described tissue of obtained precipitation is thin
The total serum IgE of born of the same parents' sample.
Preferably, when the amount of the histocyte sample is fewer, add glycogen and promote RNA to separate out.
Further, to be extracted for DNA, extracted from the middle level and organic phase, and S4 is specifically included：
S421：The middle level and organic phase are taken, moisture therein is removed, and adds straight alcohol thereto, DNA is separated out；
S422：Precipitated after centrifugation, wash centrifugation with 0.1M sodium citrate solutions and straight alcohol successively, obtained precipitation
Containing the ethanol that volume fraction is 10% in DNA in as described histocyte sample, the sodium citrate solution, and pH
For 8.5.
Further, what is extracted is protein, and S4 is specifically included：
S431：The middle level and organic phase are taken, moisture therein is removed, and adds straight alcohol thereto, DNA is separated out；
S432：After centrifugation, supernatant is taken, isopropanol is added thereto, protein is separated out；
S433：Centrifugation is precipitated, and washs centrifugation with 0.3M guanidine hydrochloride solution and straight alcohol successively, obtained precipitation is i.e.
For the protein in the histocyte sample, the solvent of the guanidine hydrochloride solution is the ethanol solution that volume fraction is 95%.
The nucleic acid-protein extracts reagent and method extraction efficiency of the present invention is high, and the integrality for obtaining nucleic acid and protein is good,
And extracting method is simple and convenient, contaminated samples are difficult, also will not be to human body occurrence injury.
Brief description of the drawings
The Ago-Gel of the total serum IgE for the mouse lung that Fig. 1 extracts for the nucleic acid-protein extracts reagent and TRIzol of the present invention
Electrophoretogram, wherein swimming lane 1 are the RNA that TRIzol is extracted, and swimming lane 2 is the RNA that nucleic acid-protein extracts reagent is extracted；
The Ago-Gel of the total serum IgE for the Mouse Kidney that Fig. 2 extracts for the nucleic acid-protein extracts reagent and TRIzol of the present invention
Electrophoretogram, wherein swimming lane 1 are the RNA that TRIzol is extracted, and swimming lane 2 is the RNA that nucleic acid-protein extracts reagent is extracted；
The agarose of the total serum IgE for the mouse muscle that Fig. 3 extracts for the nucleic acid-protein extracts reagent and TRIzol of the present invention coagulates
Gel electrophoresis figure, wherein swimming lane 1 are the RNA that TRIzol is extracted, and swimming lane 2 is the RNA that nucleic acid-protein extracts reagent is extracted.
Embodiment
The principle and feature of the present invention are described below in conjunction with example, the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the present invention.
The method for extracting nucleic acid-protein using the nucleic acid-protein extracts reagent of the present invention：
1. nucleic acid-protein extracts reagent handles sample
1) every 50~100mg sample tissue adds 1ml nucleic acid-protein extracts reagent, and crushing instrument with histocyte crushes
Afterwards, it is stored at room temperature 5~6 minutes；
2) ratio for adding 200ul chloroforms in every milliliter of nucleic acid-protein extracts reagent adds chloroform to above-mentioned homogenised sample
In；
3) sample cell is acutely overturned up and down, is mixed sample, is continued 15 seconds；
4) it is stored at room temperature the sample of mixing 2-3 minutes；
5) 4 DEG C of sample is centrifuged 15 minutes with 12000g；
6) upper strata aqueous phase (containing RNA) is taken to the further separation that RNA is carried out to new centrifuge tube；It is careful not in shake-up
Interbed and lower floor's organic phase.
7) intermediate layer and organic phase carry out the further separation of DNA and protein.
2.RNA extraction
1) (it is less than 1 × 10 when the sample size of processing is smaller6Cell or less than 10mg tissues) when, 5- will will be entered in supernatant
10 μ g's helps RNA precipitate without RNase glycogens；
2) the 500ul isopropanol of ratio addition 100% is added according to every milliliter of nucleic acid-protein extracts reagent above-mentioned
In aqueous phase；
3) room temperature is acted on 10 minutes；
4) 4 DEG C of sample is centrifuged 10 minutes with 12000g；
5) supernatant is removed；
6) ratio for adding 1ml according to every milliliter of nucleic acid-protein extracts reagent adds 75% ethanol；
7) centrifuge tube is reverse twice, 4 DEG C are centrifuged 5 minutes with 7,500g；
8) will the appropriate drying of precipitation；
9) precipitation is resuspended without RNase water or 0.5%SDS solution with 20-50 μ l；
10) water-bath or heat block are acted on 10-15 minutes in 55-60 DEG C；
11) sample can carry out subsequent applications or be stored in -70 DEG C of
3.DNA extraction：
1) moisture on intermediate layer and organic phase is fully removed；
2) ratio for adding 300ul according to every milliliter of nucleic acid-protein extracts reagent adds 100% ethanol in above-mentioned sample cell
In；
3) turn upside down mixing sample；
4) the sample room temperature after mixing is placed 2-3 minutes；
5) 4 DEG C centrifuge 5 minutes precipitation DNA with 2000g；
6) phenol-ethanol supernatant is fetched into new centrifuge tube, carries out the separation of protein；
7) ratio that precipitation adds 1ml according to every milliliter of nucleic acid-protein extracts reagent adds 0.1M sodium citrate (10% second
Alcohol is prepared, pH8.5)；
8) room temperature is acted on 30 minutes, during which suitably gently overturns centrifuge tube with biased sample；
9) 4 DEG C are centrifuged 5 minutes with 2000g, remove supernatant；
10) repeat step 7-9 processes 1 time, is repeated 2 times when DNA sample amount is more than 200 μ g；
11) ratio for adding 1.5-2ml according to every milliliter of nucleic acid-protein extracts reagent adds 75% ethanol；
12) room temperature is acted on 10-20 minutes, during which suitably gently overturns centrifuge tube with biased sample；
13) 4 DEG C are centrifuged 5 minutes with 2000g, remove supernatant；
14) DNA sample is suitably dried up；
15) according to every 50-70mg tissue samples or 1 × 107The ratio that cell concentration adds 300-600 μ l adds 8mM's
NaOH, is resuspended DNA；
16) 4 DEG C are centrifuged 10 minutes with 12000g, and supernatant moves to new centrifuge tube, remove insoluble matter；
17) supernatant is handled or preserved according to subsequent applications.
3. the extraction of protein：
The phenol taken out in above-mentioned DNA separation processes-ethanol supernatant carries out Separation of Proteins.
1) ratio for adding 1.5 milliliters of isopropanols according to every milliliter of nucleic acid-protein extracts reagent adds in phenol-ethanol supernatant
Enter isopropanol；
2) it is stored at room temperature 10 minutes after mixing；
3) 4 DEG C of sample centrifuges 10 minutes precipitating proteins with 12000g, removes supernatant；
4) protein of precipitation carries out washing step；
5) according to the ratio of every milliliter of 2 milliliters of nucleic acid-protein extracts reagent addition, adding 0.3M guanidine hydrochlorides, (95% ethanol is matched somebody with somebody
System)；
6) room temperature is acted on 20 minutes；
7) 4 DEG C of sample is centrifuged 5 minutes with 7500g, removes washing supernatant；
8) 5-7 steps, 2 times or more are repeated；
9) 2ml 100% ethanol is added in the albumen precipitation of last time washing and is vibrated；
10) room temperature is acted on 20 minutes；
11) 4 DEG C of sample is centrifuged 5 minutes with 7500g, removes ethanol supernatant；
12) deposit sample is air-dried, but without completely dry；
13) protein is resuspended the SDS solution 200ul for adding 1%, and 50 DEG C of water need to be carried out by sample by being completely dissolved protein
Bath；
14) 4 DEG C of sample is centrifuged 10 minutes with 10000g, removes insoluble matter；
15) supernatant is moved into new pipe and carries out follow-up required application.
4.RNA's quantifies：
1) sample is suitably diluted with without RNase water, and measures 260nm and 280nm absorption values.
2) formula A is utilized260× dilution factor × 40=ugRNA/ML draws concentration
5.DNA's quantifies：
1) samples with water or pH>7.5 buffer solutions suitably dilute, and measure 260nm and 280nm absorption value
2) formula A is utilized260× dilution factor × 50=ugRNA/ML draws concentration
6. protein is quantified：
Can be according to Bradford methods, SDS concentration when noting method measurement in sample is no more than 0.1%.
7. the comparison with the commodity on sale of in the market
With the lung, kidney and muscle of nucleic acid-protein extracts reagent and in the market TRIzol on sale of the invention respectively to mouse
Tissue extraction RNA, its comparative result is as shown in table 1 and Fig. 1,2 and 3, it was found from agarose gel electrophoresis figure, and two kinds of reagents are extracted
RNA it is all very complete, measure RNA concentration, nucleic acid-protein extracts reagent of the invention extracts obtained RNA concentration ratios TRIzol
It is high.
The comparison for the RNA that the nucleic acid-protein extracts reagent and TRIzol of the present invention of table 1 is extracted
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc. should be included in the scope of the protection.
Claims (9)
1. a kind of nucleic acid-protein extracts reagent, it is characterised in that the sodium chloride comprising 0.1-0.8M, 0.3-1.0M ammonium sulfate,
0.7-1.5M guanidine hydrochloride and volume fraction 10-50% phenol.
2. application of the nucleic acid-protein extracts reagent in nucleic acid and/or Protein Extraction described in claim 1.
3. one kind extracts nucleic acid and/or method of protein from histocyte sample, it is characterised in that comprise the following steps：
S1：By the histocyte sample broke into homogenate；
S2：The organic solvent diluting liquid of the nucleic acid-protein extracts reagent described in claim 1 is added into the homogenate, is mixed,
Obtain extracting mixed liquor；
S3：The extraction mixed liquor is centrifuged, layering obtains aqueous phase, middle level and organic phase；
S4：The nucleic acid and/or protein of the histocyte sample are extracted from corresponding layer.
4. method according to claim 3, it is characterised in that the histocyte sample is extracted with the nucleic acid-protein and tried
The ratio of the amount of agent is per nucleic acid-protein extracts reagent described in 50-100mg histocytes sample addition 1ml.
5. method according to claim 3, it is characterised in that the organic solvent is chloroform, and the nucleic acid-protein
Extracts reagent and the volume ratio of chloroform are 1：0.1-0.5.
6. the method according to any one of claim 3-5, it is characterised in that to be extracted for RNA, from the aqueous phase
It is middle to extract, and S4 specifically includes：
S411：The aqueous phase in S3 is taken, adding isopropanol separates out RNA；
S412：Precipitated after centrifugation, centrifugation is washed with the ethanol of volume fraction 75%, the i.e. described histocyte sample of obtained precipitation
The total serum IgE of product.
7. method according to claim 6, it is characterised in that in S411, adds glycogen promotion RNA also into the aqueous phase
Separate out.
8. the method according to any one of claim 3-5, it is characterised in that to be extracted for DNA, from the middle level
Extracted with organic phase, and S4 is specifically included：
S421：The middle level and organic phase are taken, moisture therein is removed, and adds straight alcohol thereto, DNA is separated out；
S422：Precipitated after centrifugation, wash centrifugation with 0.1M sodium citrate solutions and straight alcohol successively, obtained precipitation is
Containing the ethanol that volume fraction is 10% in DNA in the histocyte sample, the sodium citrate solution, and pH is
8.5。
9. the method according to any one of claim 3-5, it is characterised in that what is extracted is protein, and S4
Specifically include：
S431：The middle level and organic phase are taken, moisture therein is removed, and adds straight alcohol thereto, DNA is separated out；
S432：After centrifugation, supernatant is taken, isopropanol is added thereto, protein is separated out；
S433：Centrifugation is precipitated, and washs centrifugation with 0.3M guanidine hydrochloride solution and straight alcohol successively, obtained precipitation as institute
The protein in histocyte sample is stated, the solvent of the guanidine hydrochloride solution is the ethanol solution that volume fraction is 95%.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111269284A (en) * | 2018-12-04 | 2020-06-12 | 中国科学院大连化学物理研究所 | Reagent and method for synchronously separating protein and RNA in cytoplasm and nucleus |
| CN111592586A (en) * | 2019-11-24 | 2020-08-28 | 杭州市农业科学研究院 | Strawberry pistil protein extraction method suitable for mass spectrum identification |
| CN115532242A (en) * | 2022-10-18 | 2022-12-30 | 广州美基生物科技有限公司 | Humic acid adsorbent and preparation method thereof soil DNA extraction kit |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103080311A (en) * | 2010-09-06 | 2013-05-01 | 恰根有限公司 | Method of isolating purified RNA with reduced DNA contaminations |
| CN105368816A (en) * | 2015-11-25 | 2016-03-02 | 湖北省农业科学院畜牧兽医研究所 | Method for separating DNA, RNA and protein from single plant sample |
-
2017
- 2017-03-03 CN CN201710124470.2A patent/CN106978414A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103080311A (en) * | 2010-09-06 | 2013-05-01 | 恰根有限公司 | Method of isolating purified RNA with reduced DNA contaminations |
| CN105368816A (en) * | 2015-11-25 | 2016-03-02 | 湖北省农业科学院畜牧兽医研究所 | Method for separating DNA, RNA and protein from single plant sample |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111269284A (en) * | 2018-12-04 | 2020-06-12 | 中国科学院大连化学物理研究所 | Reagent and method for synchronously separating protein and RNA in cytoplasm and nucleus |
| CN111269284B (en) * | 2018-12-04 | 2023-07-25 | 中国科学院大连化学物理研究所 | Reagents and methods for simultaneous separation of proteins and RNAs in cytoplasm and nucleus |
| CN111592586A (en) * | 2019-11-24 | 2020-08-28 | 杭州市农业科学研究院 | Strawberry pistil protein extraction method suitable for mass spectrum identification |
| CN115532242A (en) * | 2022-10-18 | 2022-12-30 | 广州美基生物科技有限公司 | Humic acid adsorbent and preparation method thereof soil DNA extraction kit |
| CN115532242B (en) * | 2022-10-18 | 2023-05-26 | 广州美基生物科技有限公司 | Humic acid adsorbent and preparation method thereof as well as soil DNA extraction kit |
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Application publication date: 20170725 |