CN109576262A - A kind of preservation formula of liquid being widely used in plant field sampling and its application method - Google Patents
A kind of preservation formula of liquid being widely used in plant field sampling and its application method Download PDFInfo
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- CN109576262A CN109576262A CN201710893763.7A CN201710893763A CN109576262A CN 109576262 A CN109576262 A CN 109576262A CN 201710893763 A CN201710893763 A CN 201710893763A CN 109576262 A CN109576262 A CN 109576262A
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- 238000004321 preservation Methods 0.000 title claims abstract description 12
- 238000005070 sampling Methods 0.000 title claims abstract description 12
- 239000007788 liquid Substances 0.000 title claims description 15
- 238000000034 method Methods 0.000 title description 11
- 229920001817 Agar Polymers 0.000 claims abstract description 11
- 239000008272 agar Substances 0.000 claims abstract description 11
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 6
- 239000000843 powder Substances 0.000 claims abstract description 6
- 238000000605 extraction Methods 0.000 claims abstract description 4
- 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 3
- 102100030310 5,6-dihydroxyindole-2-carboxylic acid oxidase Human genes 0.000 claims abstract description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 3
- 101000773083 Homo sapiens 5,6-dihydroxyindole-2-carboxylic acid oxidase Proteins 0.000 claims abstract description 3
- SZHJTBRHSAKQIF-UHFFFAOYSA-N [Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].OB(O)O.[O-]B([O-])[O-].[O-]B([O-])[O-].[O-]B([O-])[O-].[O-]B([O-])[O-] Chemical compound [Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].OB(O)O.[O-]B([O-])[O-].[O-]B([O-])[O-].[O-]B([O-])[O-].[O-]B([O-])[O-] SZHJTBRHSAKQIF-UHFFFAOYSA-N 0.000 claims abstract description 3
- 239000008103 glucose Substances 0.000 claims abstract description 3
- 239000011780 sodium chloride Substances 0.000 claims abstract description 3
- 241000196324 Embryophyta Species 0.000 claims description 16
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 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 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 2
- 239000008223 sterile water Substances 0.000 claims description 2
- 238000011282 treatment Methods 0.000 claims description 2
- 150000007523 nucleic acids Chemical class 0.000 abstract description 4
- 102000039446 nucleic acids Human genes 0.000 abstract description 4
- 108020004707 nucleic acids Proteins 0.000 abstract description 4
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 abstract 1
- 239000002360 explosive Substances 0.000 abstract 1
- 239000000284 extract Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000002798 spectrophotometry method Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 1
- 241000219000 Populus Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229960004756 ethanol Drugs 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000011221 initial treatment Methods 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000012372 quality testing Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- -1 which is added Substances 0.000 description 1
Classifications
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- 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
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Saccharide Compounds (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to field of plant molecular biology, are related to a kind of preservation formula for being widely used in plant field sampling.It is characterized in that being made of following component and its content: component A: 50mM Tris-HCl (PH=8.0), 1mM Na2EDTA, 100mM glucose, 5% glycerol, boric acid-sodium tetraborate buffer 0.025mM, NaCl 1.6mM, CATB 0.5% (m/v)；B component: agar powder.The present invention can guarantee extraneous no more than 50 DEG C, time is no more than the integrality that can completely save any plant sample RNA in the case where 72h, and the solution will not generate obvious smell, it is harmless, during transportation also without any harm such as inflammable and explosive, the general purpose vehicles such as train of getting on the car can be carried, can also be transported by modes such as aircrafts.After returning to laboratory, the nucleic acid extraction in sample can be come out by special extracting solution, Nucleic acid quality, which can achieve, builds library standard.
Description
Technical field:
The invention belongs to field of plant molecular biology, are related to a kind of solid conservation for being widely used in plant field sampling
Formula.
Background technique:
It is highly important means in plant subject, on this basis, chip analysis from the variation of Molecular level study plant
And the technologies such as high-flux sequence bring very huge data information to us.And at present the trouble that encounters of people first is that:
Many plant samples are located remotely from the place in laboratory, such as remote wild life conservation, the mistake drawn materials in this way
Journey at least needs following steps: excessively remote place uses manual sampling, and material is carried out primary treatment in sample point
Afterwards, it is advantageously local to be transported to traffic, carries out sample transfer until laboratory by the vehicles.And nucleic acid, especially
It is Ribonucleic RNA, is degraded in the time of in vitro 0.2-0.5h or so, even if some materials can be stored in ethyl alcohol etc. and have
In solvent, the same larger and transportational process safety of loss amount is poor during transportation.Therefore plant tissue is transported and is saved
The research of method is still improved space.
Summary of the invention:
The purpose of the invention is to provide a kind of preservation formulas of plant field sampling, pass through this plant field sampling
The use of liquid is saved, got sample is protected not come to harm, in scientific research field, is provided complete required for experiment
RNA。
The technical scheme is that
The preservation formula of liquid of 1 plant field sampling, it is characterized in that being made of following component and its content: component A: 50mM
Tris-HCl (PH=8.0), 1mM EDTA, glucose 100mM, glycerol 3%, boric acid-sodium tetraborate buffer 0.025mM,
NaCl 1.6mM, CATB 0.5% (m/v)；B component: agar powder 15g/l.It is dissolved through sterile water and suitably heats (50-60 degree)
It is completely melt to agar powder, plant specimen is submerged after liquid is cooled to 40 degree and is used.
The preservation liquid laboratory treatment mode of 2 plant field samplings: agar around sample is removed, by sample and residual fine jade
Rouge is put into together in 50ml centrifuge tube, and the mixing of 3mM guanidinium isothiocyanate is added, and saves liquid by every ml and 0.05gSiO is added2, 55 degree
Water-bath 10min acutely shakes 15min with oscillator after grinding 5min with grinding rod after taking-up.12000rpm is centrifuged 5min, will
Supernatant is carefully transferred in another centrifuge tube, and isometric chloroform is added and is shaken, finishing operations are consistent with general extraction methods.
Detailed description of the invention:
Fig. 1 is saved through this product and fresh sample directly extracts total RNA content comparison diagram
Fig. 2 RNA Quality Map
1 spectrophotometry result of table
Specific embodiment:
1. field sampling: 50ml being saved in liquid (A ingredient), 0.75g agar powder (B component) is added, placed after heating for dissolving
It is cooling on one side；Collected animals and plants sample is put into 50ml centrifuge tube and (is put into centrifuge tube after being cut if necessary
In), it pours into centrifuge tube rapidly when the above-mentioned preservation liquid heated is cooled to 40 degree or less, uses nozzle after being cooled to solid
Parafilm sealed membrane is completely enclosed.
2. it should be noted that preventing the pollution to pipe such as high temperature (being greater than 50 degree) or rainwater in transportational process.Transportational process Ying Buchao
Cross 120h.
3. laboratory pre-processes: above-mentioned solid conservation block being taken out from centrifuge tube, removes extra agar sections, by sample
Product and residual agar are put into together in new centrifuge tube, and the mixing of 3mol/l guanidinium isothiocyanate is added, and are saved liquid by every ml and are added
0.05gSiO2, 55 degree of water-bath 10min, after taking-up with grinding rod grind 5min, acutely shake 15min with oscillator later.
12000rpm is centrifuged 5min, and supernatant is carefully transferred in another centrifuge tube, isometric chloroform is added and is shaken.
4. 4 DEG C of 13000rpm are centrifuged 8min, supernatant is taken.It is repeated once above-mentioned steps.
5. supernatant, which is added, precipitates 15min, 13000rpm centrifugation with 1/10 volume NaAc and diploid product dehydrated alcohol
10min.75% alcohol, which is washed, to be dissolved in twice in the water (DEPC water) of 30ul.
Implementation result:
Extraction efficiency detection: same weight sample is divided into two groups, first group uses traditional method for extracting；Second of use
Homemade preservation liquid extracts after carrying out preservation 72h, is detected by spectrophotometry to nucleic acid.The result shows that
The sample nucleic total amount that liquid saves, which is saved, through self-control does not substantially reduce that (Fig. 1 is protected through this product compared with traditional method for extracting
It deposits and fresh sample directly extracts total RNA content comparison diagram).
It extracts quality testing: same weight sample being divided into two groups, first group uses traditional method for extracting；Second of use
Homemade preservation formula extracts after carrying out preservation 72h, the results showed that, RNA degradation does not all occur for two groups of extracted samples
(Fig. 2 RNA Quality Map: left side swimming lane is that fresh sample extracts RNA to phenomenon, and right side is shown as after this product saves 72h and extracts
RNA is as a result, sample is poplar leaf, applied sample amount 3ul), with spectrophotometry to extracted 230,260,280 He of sample
320nm wavelength absorption photometric value is detected, and two groups of sample results are good, and capillary electrophoresis testing result is shown, self-control is protected
Storage sample this RIN value is greater than 8, indicates that RNA integrality is good, fully meets and build the biological experiments such as library needs (table 1).
1 spectrophotometry result of table
Claims (2)
1. the solid conservation formula of liquid of plant field sampling, it is characterized in that being made of following component and its content: component A: 50mM
Tris-HCl (PH=8.0), 1mM EDTA, glucose 100mM, glycerol 3%, boric acid-sodium tetraborate buffer 0.025mM,
NaCl 1.6mM, CATB 0.5% (m/v)；B component: agar powder 15g/l.It is dissolved through sterile water and suitably heats (40-50 degree)
It is completely melt to agar powder and uses after submerging plant specimen.
2. the preservation liquid laboratory treatment mode of plant field sampling: agar around sample being removed, by sample and residual agar
It is put into centrifuge tube together, the mixing of 3mM guanidinium isothiocyanate is added, save liquid by every ml and 0.05g SiO is added2, 55 degree of water-baths
10min acutely shakes 15min with oscillator after grinding 5min with grinding rod after taking-up.12000rpm is centrifuged 5min, by supernatant
It is carefully transferred in another centrifuge tube, isometric chloroform is added and is shaken, finishing operations are consistent with general extraction methods.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710893763.7A CN109576262A (en) | 2017-09-28 | 2017-09-28 | A kind of preservation formula of liquid being widely used in plant field sampling and its application method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710893763.7A CN109576262A (en) | 2017-09-28 | 2017-09-28 | A kind of preservation formula of liquid being widely used in plant field sampling and its application method |
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| Publication Number | Publication Date |
|---|---|
| CN109576262A true CN109576262A (en) | 2019-04-05 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201710893763.7A Pending CN109576262A (en) | 2017-09-28 | 2017-09-28 | A kind of preservation formula of liquid being widely used in plant field sampling and its application method |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102805078A (en) * | 2012-08-20 | 2012-12-05 | 中国热带农业科学院橡胶研究所 | Short-term preservation method of rubber tree leaves for RNA (ribonucleic acid) extraction |
| CN103820320A (en) * | 2013-06-25 | 2014-05-28 | 宁波有成生物医药科技有限公司 | Non-freezing type RNA (Ribonucleic Acid) protection fluid |
| CN104152439A (en) * | 2014-08-30 | 2014-11-19 | 中国烟草总公司郑州烟草研究院 | RNA extraction method suitable for tobacco seeds |
| CN104404007A (en) * | 2014-11-06 | 2015-03-11 | 中国热带农业科学院海口实验站 | Banana polyphenol oxidase gene, recombinant protein, and preparation method thereof |
| CN104673717A (en) * | 2015-02-12 | 2015-06-03 | 中国医科大学 | Biological tissue RNA (ribonucleic acid) protection reagent as well as preparation method and application thereof |
| CN105506129A (en) * | 2016-01-01 | 2016-04-20 | 广州邦德盛生物科技有限公司 | RNA type sample preservation and dilution liquid and preparation thereof |
-
2017
- 2017-09-28 CN CN201710893763.7A patent/CN109576262A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102805078A (en) * | 2012-08-20 | 2012-12-05 | 中国热带农业科学院橡胶研究所 | Short-term preservation method of rubber tree leaves for RNA (ribonucleic acid) extraction |
| CN103820320A (en) * | 2013-06-25 | 2014-05-28 | 宁波有成生物医药科技有限公司 | Non-freezing type RNA (Ribonucleic Acid) protection fluid |
| CN104152439A (en) * | 2014-08-30 | 2014-11-19 | 中国烟草总公司郑州烟草研究院 | RNA extraction method suitable for tobacco seeds |
| CN104404007A (en) * | 2014-11-06 | 2015-03-11 | 中国热带农业科学院海口实验站 | Banana polyphenol oxidase gene, recombinant protein, and preparation method thereof |
| CN104673717A (en) * | 2015-02-12 | 2015-06-03 | 中国医科大学 | Biological tissue RNA (ribonucleic acid) protection reagent as well as preparation method and application thereof |
| CN105506129A (en) * | 2016-01-01 | 2016-04-20 | 广州邦德盛生物科技有限公司 | RNA type sample preservation and dilution liquid and preparation thereof |
Non-Patent Citations (2)
| Title |
|---|
| MIGUEL CAMACHO-SANCHEZ 等: "Preservation of RNA and DNA from mammal samples under field conditions", 《MOLECULAR ECOLOGY RESOURCES》 * |
| 史公军 等: "白菜花药组织总RNA提取方法比较及其分析", 《西北农业学报》 * |
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Application publication date: 20190405 |