CN102140451A - Method for extracting DNA (Desoxyribonucleic Acid) and RNA (Ribonucleic Acid) - Google Patents
Method for extracting DNA (Desoxyribonucleic Acid) and RNA (Ribonucleic Acid) Download PDFInfo
- Publication number
- CN102140451A CN102140451A CN2011100092550A CN201110009255A CN102140451A CN 102140451 A CN102140451 A CN 102140451A CN 2011100092550 A CN2011100092550 A CN 2011100092550A CN 201110009255 A CN201110009255 A CN 201110009255A CN 102140451 A CN102140451 A CN 102140451A
- Authority
- CN
- China
- Prior art keywords
- dna
- rna
- cracking
- precipitation
- digestion product
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a method for extracting DNA (Desoxyribonucleic Acid) and RNA (Ribonucleic Acid) from prokaryotic or eukaryotic materials, comprising the following steps: (1) pretreatment: cracking prokaryotic or eukaryotic materials by liquid nitrogen grinding method, or by repeated blowing-sucking method with liquid-moving machine suction head, or by glass bead method, and then carrying out dissolving treatment by using decontaminating reagents to obtain a cracked digestion product; (2) adding saturated saline solution to the cracked digestion product and then carrying out protein precipitation for the biological materials; and (3) separating precipitation phase from liquid phase, and then separating and purifying the DNA or RNA in the liquid phase. Compared with prior art, the invention has the advantages that (1) non-toxicity reagent is used; (2) the extraction method is convenient and rapid; (3) the obtained DNA or RNA has high quality and is completely suitable for downstream molecular biology experiments; and (4) the cost is low.
Description
Technical field
The present invention relates to biotechnology, specifically, relate to the method for a kind of DNA of extraction and RNA.
Background technology
Along with biotechnology development with rapid changepl. never-ending changes and improvements, the also increasingly mature and development of the extractive technique of DNA and RNA, at present existing a large amount of technology is widely used in laboratory, industrial production, pharmacy and Animal or Plant Quarantine field, also produced a large amount of relevant patents, reached " method of isolating nucleic acid " (200680034578.2) etc. as " a kind of magnetic nano particle compound and method for making and application (200610023144.4) that is used for separate nucleic acid ".
DNA and RNA always combine with range protein in cell.Mainly be meant the separating of DNA and RNA DNA and RNA are separated with biomacromolecule materials such as protein, polysaccharide, fat.When DNA isolation and RNA, should guarantee the integrity of DNA and RNA molecule primary structure, get rid of other molecular contaminations.But owing to contain carbohydrate, tannic acid, polyphenol and albumen because DNA and RNA extract in the sample, even the organic reagent such as phenol and the chloroform that use in extracting, how therefore the capital contaminated samples obtain highly purified DNA and RNA, just becomes the problem that all investigators have to face.
The method that the purpose of this invention is to provide a kind of DNA of extraction and RNA.
Summary of the invention
The purpose of this invention is to provide and a kind ofly from protokaryon or eukaryote source material, extract nucleic acid, be i.e. the method for DNA or RNA.
The electronegative characteristic of nucleic acid molecule phosphate group of utilization of the present invention is developed, and uses in the positive charge in the saturated aqueous common salt and the negative charge of above-mentioned phosphate group, and precipitate and separate is passed through purifying at last then, obtains DNA or RNA.
Specifically, the method for extraction DNA of the present invention or RNA comprises the steps:
1) pre-treatment: cracking protokaryon or eukaryote material also carry out dissolution process with stain remover, obtain the cracking digestion product;
2) saturated aqueous common salt is joined in the cracking digestion product, the albumen in the biomaterial is carried out precipitation process;
3) precipitation separation mutually and liquid phase, then to DNA in the liquid phase or RNA separates and purifying.
Wherein, step 2) described salt solution is neutral or acid saturated brine, if needs extraction DNA then uses neutral saturated brine; If need to extract RNA, then use the acid saturated brine of pH value 5-6;
Wherein, the volumetric usage of described saturated aqueous common salt be cracking digestion product volume 0.3-1 doubly;
Described neutral saturated brine is NaCl directly to be dissolved in boil preparation in the pure water; Described acid saturated brine adds hydrochloric acid to neutral saturated brine and obtains, and makes the pH value reach 5-6.
Step 2) following carrying out: the neutral saturated salt solution that in every milliliter cracking digestion product, adds 350 μ l earlier; Then above-mentioned solution is put upside down mixing gently, at room temperature place 5-15min then;
Step 3) can be carried out according to technology well known in the art, for example can followingly carry out: above-mentioned solution at the centrifugal 15-30min of room temperature 2500rpm, must be contained the supernatant liquor of DNA or RNA, adopt following method for containing the DNA supernatant liquor then:
1. deposit D NA
1.1 shift the extremely new Eppendorf pipe of supernatant that contains DNA;
1.2 the centrifugal 10min of room temperature 12000rpm, supernatant discarded;
2. wash DNA
2.1DNA precipitation adds 75% ethanol;
2.2 the centrifugal 5min of room temperature 10000rpm, supernatant discarded;
3. dissolving DNA
3.1 dry DNA precipitation 5min is dissolved in the ultrapure water then under the room temperature condition;
3.2 being carried out quantitatively also packing, the DNA that obtains is stored in-80 ℃.
4. remove the RNA pollutent
4.1 add the RNase of 50 μ g in every milliliter the dna solution;
4.2 37 ℃ of incubation 1h.
Annotate: removal RNA pollutes employed RNase facture can only be used in the final step that extracts.Desalination obtains high-quality DNA if desired, and RNase handles the step 3 and 4 of available the methodology in back and carries out.If will preserve DNA for a long time, available Tris-EDTA (TE) solution dissolving DNA is to suppress nuclease.
Adopt following method for containing the RNA supernatant liquor:
1.RNA precipitation
1.1 shift the extremely new Eppendorf pipe of supernatant that contains RNA;
1.2 the centrifugal 10min of room temperature 12000rpm, supernatant discarded;
2.RNA washing
2.1RNA precipitation adds 75% ethanol;
2.2 the centrifugal 5min of room temperature 10000rpm, supernatant discarded;
3.RNA dissolving
3.1 dry RNA precipitation 5min is dissolved in the ultrapure water after DEPC handles then under the room temperature condition;
3.2 being carried out quantitatively also packing, the RNA that obtains is stored in-80 ℃.
4. remove the genomic dna pollutent
The available DNase I of this part (no RNase) test kit is operated, and concrete steps are referring to the specification sheets of manufacturers.
In step 1), different according to the source of biomaterial and quantity, described pre-treatment can followingly be carried out:
1), adopts the liquid nitrogen grinding method and carry out dissolution process with stain remover subsequently for comparatively a large amount of biomaterials;
2) for single zooblast or bacterial cultures, employing pipettor suction nozzle pressure-vaccum method repeatedly also carries out dissolution process with stain remover subsequently;
3) for comprising parasitic ovum, the other biological material of plant etc. can adopt the granulated glass sphere method of breaking also to carry out dissolution process with stain remover subsequently.
Below pretreatment process is described in detail:
1) the liquid nitrogen grinding method is extracted DNA or RNA:
1.1 get tissue to be measured, chopping is placed on mortar;
1.2 add liquid nitrogen with frozen tissue;
1.3 use pestle tissue abrasion;
1.4 every g tissue to be measured adds the lysate of 3ml;
1.5 every g tissue to be measured adds 10% SDS of 300 μ l;
1.6 abrasive material is transferred to the Eppendorf pipe;
1.7 the adding Proteinase K digests 1h at 50 ℃;
2) pipettor repeatedly the pressure-vaccum method extract DNA or RNA (is example with colibacillary nucleic acid extraction)
1.1 bacterial cultures or cell culture are transferred in the Eppendorf pipe, and the centrifugal 3-10min of 2000rpm obtains precipitation
1.2 supernatant discarded, add lysate in the precipitation and with pipettor suction nozzle pressure-vaccum 8-10 time with cracking thalline or cell;
1.3 every milliliter of bacterial cultures adds 100 μ l, 10% SDS;
1.4 every milliliter of lysate adds 100 μ g Proteinase Ks, incubation 1h in 50 ℃ of water-baths.
3) the granulated glass sphere crush method is extracted DNA or RNA
1.1 biomaterial is mixed concussion 10min fragmentation with the sterilization granulated glass sphere;
1.2 every milliliter adds 300 μ l, 10% SDS;
1.3 every milliliter of lysate adds 300 μ g Proteinase Ks, then at 50 ℃ of incubation 1h ℃.
Wherein, described sterilization granulated glass sphere is selected this area granulated glass sphere commonly used, preferred 0.4mm diameter.Described concussion adopts high speed vortex oscillation device to carry out, and its rotating speed is 5000rpm.
Core of the present invention be with in the positively charged ion of salts solution and nucleic acid with negative charge, DNA or RNA after being neutralized then separate out in high level salt solution.Method by low-speed centrifugal can be removed most of impurity such as cell debris, albumen, DNA or RNA can be precipitated out by the ethanol sedimentation rule.Compared with prior art, the invention has the advantages that: 1) use nontoxic reagent; 2) simple and convenient extraction is quick; 3) with low cost.
Description of drawings
Fig. 1. use the genomic dna (first and second swimming lanes) of the precocious Eimeria (Eimeria praecox) of method extraction of the present invention.Dna molecular amount mark (M) is the λ genome of EcoT14 enzyme after cutting.
Fig. 2. use the genomic dna of the Eimeria tenella (Eimeria tenella) of method extraction of the present invention.The dna molecular amount is labeled as the λ genome after the EcoT14 enzyme is cut.
Fig. 3. use total RNA of the precocious Eimeria (Eimeria praecox) of method extraction of the present invention.The dna molecular amount is labeled as Trans DNA Marker III.
Fig. 4. total RNA of the bold and unconstrained strain (Hougton Strain) of pausing of the Eimeria tenella (Eimeria tenella) of using method of the present invention to extract.The dna molecular amount is labeled as Trans DNA Marker III.
The MIC2 gene that successfully increases and obtain behind the genome of the bold and unconstrained strain (Hougton Strain) of the Eimeria tenella (Eimeria tenella) that Fig. 5 uses method of the present invention to extract and Beijing strain (Beijing Strain).
Fig. 6. use the genomic dna of the BALB/c mouse of method extraction of the present invention.Dna molecular amount mark (M) is the λ genome of EcoT14 enzyme after cutting.
The 16S rDNA gene that Fig. 7 uses behind the genome of the mouse that method of the present invention extracts successfully amplification to obtain.
Fig. 8. the colibacillary genomic dna that uses method of the present invention to extract.Dna molecular amount mark (M) is the λ genome of EcoT14 enzyme after cutting.
Colibacillary total RNA that Fig. 9 uses method of the present invention to extract.The dna molecular amount is labeled as Trans DNA Marker III.
Figure 10. the 16S rDNA gene that successfully increases and obtain behind the colibacillary genome that uses method of the present invention to extract.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Utilize the granulated glass sphere crush method to extract the DNA or the RNA of Eimeria egg capsule sample, operation steps is as follows:
One, DNA extraction operation scheme
1. cytoclasis:
1.1 5 * 10
6Individual sporeization or not the egg capsule precipitation of sporeization mixes with the sterilization granulated glass sphere (0.4mm diameter) of 0.5g, in the screw-cap centrifuge tube of the sharp end of the 15ml that packs into, shake 10min fragmentation egg capsule with high speed vortex oscillation device (5000rpm);
1.2 every milliliter adds 300 μ l 10%SDS;
1.3 add 300 μ g Proteinase Ks, then at 50 ℃ of incubation 1h.
2. be separated:
2.1 add the neutral saturated salt solution NaCl of 1ml in the above-mentioned split product;
2.2 centrifuge tube is put upside down mixing 15s gently, at room temperature places 10min then;
2.3 the centrifugal 15min of 2500rpm under the room temperature;
2.4DNA molecule only is present in supernatant aqueous phase (See Figure) at this moment
3.DNA precipitation
3.1 shift the extremely new Eppendorf pipe of supernatant that contains DNA;
3.2 the centrifugal 10min of room temperature 12000rpm, supernatant discarded
1.3.DNA precipitation
4.DNA washing
4.1DNA precipitation adds 75% ethanol;
4.2 the centrifugal 5min of room temperature 10000rpm, supernatant discarded;
5.DNA dissolving
5.1 dry DNA precipitation 5min is dissolved in the ultrapure water then under the room temperature condition;
5.2 being carried out quantitatively also packing, the DNA that obtains is stored in-80 ℃.
6. remove the RNA pollutent
6.1 add the RNase of 50 μ g in every milliliter the dna solution;
6.2 37 ℃ of incubation 1h.
Annotate: removal RNA pollutes employed RNase facture can only be used in the final step that extracts.Desalination obtains high-quality DNA if desired, and RNase handles the step 3 and 4 of available the methodology in back and carries out.If will preserve DNA for a long time, available Tris-EDTA (TE) solution dissolving DNA is to suppress nuclease.
Two, RNA extracts operation scheme
1. lysis
1.1 5 * 10
6Individual sporeization or not the precipitation of spore egg capsule mix with the sterilization granulated glass sphere (0.4mm diameter) of 0.5g, in the screw-cap centrifuge tube of the sharp end of the 15ml that packs into, with the broken egg capsule of high speed vortex oscillation device (5000rpm) concussion 10min;
1.2 every milliliter of above-mentioned product adds 300 μ l 10%SDS and mixings, places 42 ℃ of incubation 1h then.
2. be separated:
2.1 the pH that adds 1ml in the above-mentioned split product is 5.6 acid saturated salt solution NaCl;
2.2 centrifuge tube is put upside down mixing 15s gently, at room temperature places 10min then;
2.3 the centrifugal 15min of 2500rpm under the room temperature;
2.4RNA molecule only is present in supernatant aqueous phase (See Figure) at this moment
3.RNA precipitation
3.1 shift the extremely new Eppendorf pipe of supernatant that contains RNA;
3.2 the centrifugal 10min of room temperature 12000rpm, supernatant discarded;
4.RNA washing
4.1RNA precipitation adds 75% ethanol;
4.2 the centrifugal 5min of room temperature 10000rpm, supernatant discarded;
5.RNA dissolving
5.1 dry RNA precipitation 5min is dissolved in the ultrapure water after DEPC handles then under the room temperature condition;
5.2 being carried out quantitatively also packing, the RNA that obtains is stored in-80 ℃.
6. then to specifications, utilize DNase I (no RNase) to remove the genomic dna pollutent
Three, DNA extraction result
The DNAA26o/A28o ratio that extracts is 1.87, and its quality meets follow-up molecular biology operational requirement fully.The output of DNA depends on the quantity of initial egg capsule, promptly 6 * 10
6Individual spore egg capsule can obtain 3.5985 mgDNA.Fig. 1 and Fig. 2 are the 1.5% agarose gel electrophoresis figure of the DNA of extraction.
Four, RNA extracts the result
The RNAA26o/A28o ratio that extracts is 1.42, and its quality meets follow-up molecular biology operational requirement fully.The output of DNA depends on the quantity of initial egg capsule, promptly 6 * 10
6Individual spore egg capsule can obtain 191.7 μ g RNA.Fig. 3 and Fig. 4 are the 1.5% agarose gel electrophoresis figure of the RNA of extraction.
Five, genomic dna of Ti Quing and RNA are in the application of downstream process
Available special primer is the specific target gene of pcr amplification from the DNA that extracts or RNA, DNA that extracts with checking or the applicability of RNA.Use MIC2-UP (tatggctcgagcgttgtcgctg) in this example and MIC2-D (gtcaggatgactgttgagtgtc) carries out the amplification of MIC2 gene (GENBANK accession number FJ807654) from the Eimeria tenella genomic dna that is extracted.Employed primer is synthesized by the biological company limited of Beijing AudioCodes.
The MIC2 gene of Fig. 5 for from the genomic dna of strain of Eimeria tenella Beijing and Hao Dun strain, increasing.
Utilize the liquid nitrogen grinding method to extract the DNA or the RNA of mouse cell, operation steps is as follows:
One liquid nitrogen grinding method is extracted the technical scheme of DNA
1. grind
1.1 get the 1g tissue, chopping is placed on mortar;
1.2 add liquid nitrogen with frozen tissue;
1.3 use pestle tissue abrasion;
1.4 add the lysate of 3ml;
1.5 add the 10%SDS of 300 μ l;
1.6 abrasive material is transferred to the Eppendorf pipe;
Digest (50 ℃ of ℃ of 1h) 1.7 add Proteinase K
2. extracting
2.1 in every milliliter cracking digestion product, add the neutral saturated salt solution (NaCl) of 350 μ l
2.2 above-mentioned solution is put upside down mixing 15s gently, at room temperature places 10min then;
2.3 the centrifugal 15min of room temperature 2500rpm, the supernatant aqueous phase then contains DNA.
3.DNA precipitation
3.1 shift the extremely new Eppendorf pipe of supernatant that contains DNA;
3.2 the centrifugal 10min of room temperature 12000rpm, supernatant discarded;
4.DNA washing
4.1DNA precipitation adds 75% ethanol;
4.2 the centrifugal 5min of room temperature 10000rpm, supernatant discarded;
5.DNA dissolving
5.1 dry DNA precipitation 5min is dissolved in the ultrapure water then under the room temperature condition;
5.2 being carried out quantitatively also packing, the DNA that obtains is stored in-80 ℃.
6. remove the RNA pollutent
6.1 add the RNase of 50 μ g in every milliliter the dna solution;
6.2 37 ℃ of incubation 1h.
Annotate: removal RNA pollutes employed RNase facture can only be used in the final step that extracts.Desalination obtains high-quality DNA if desired, and RNase handles the step 3 and 4 of available the methodology in back and carries out.If will preserve DNA for a long time, available Tris-EDTA (TE) solution dissolving DNA is to suppress nuclease.
Two. the technical scheme that liquid nitrogen grinding method RNA extracts
1. grind
1.1 get the 1g tissue, chopping is placed on mortar;
1.2 add liquid nitrogen with frozen tissue;
1.3 use pestle tissue abrasion;
1.4 add the lysate of 3ml;
1.5 add the 10%SDS of 300 μ l;
1.6 abrasive material is transferred to the Eppendorf pipe;
Digest (50 ℃ of 1h) 1.7 add Proteinase K
2. extracting
2.1 the pH that adds 350 μ l in every milliliter cracking digestion product is 6 acid saturated salt solution (NaCl)
2.2 above-mentioned solution is put upside down mixing 15s gently, at room temperature places 10min then;
2.3 the centrifugal 15min of room temperature 2500rpm, the supernatant aqueous phase then contains RNA.
3.RNA precipitation
3.1 shift the extremely new Eppendorf pipe of supernatant that contains RNA;
3.2 the centrifugal 10min of room temperature 12000rpm, supernatant discarded;
4.RNA washing
4.1RNA precipitation adds 75% ethanol;
4.2 the centrifugal 5min of room temperature 10000rpm, supernatant discarded;
5.RNA dissolving
5.1 dry RNA precipitation 5min is dissolved in the ultrapure water after DEPC handles then under the room temperature condition;
5.2 being carried out quantitatively also packing, the RNA that obtains is stored in-80 ℃.
6. remove the genomic dna pollutent
The available DNase I of this part (no RNase) test kit is operated, and concrete steps are referring to the specification sheets of manufacturers.
Three, DNA extraction effect
The DNAA26o/A28o ratio that extracts is 1.83, and its quality meets follow-up molecular biology operational requirement fully.The output of DNA depends on the amount of initial organization material, and promptly the tissue from 10mg can obtain 200 μ gDNA.Fig. 6 is the 1.5% agarose gel electrophoresis figure of the DNA of extraction.
Genomic dna that use the present invention extracts and RNA are in the application of downstream process
Available special primer is the specific target gene of pcr amplification from the DNA that extracts or RNA, DNA that extracts with checking or the applicability of RNA.Use forward GAPDH Primer (5 '-CAAGGTCATCCATGACAACTTTG-3 ' and reverse GAPDH Primer (5 '-GTCCACCACCCTGTTGCTGTAG-3 ') in this example and from the mouse gene group DNA that is extracted, carry out the amplification of 16S rDNA.Employed primer is synthesized by the biological company limited of Beijing AudioCodes.The results are shown in Figure 7.
Utilize the pipettor suction nozzle repeatedly the pressure-vaccum method extract colibacillary DNA or RNA, operation steps is as follows:
One, DNA extraction operation scheme
1 homogenate
1.1 the bacterial cultures of 1ml is transferred to the centrifugal 5min acquisition of 2000rpm precipitation in the Eppendorf pipe
1.2 supernatant discarded adds the 1ml lysate and uses pipettor suction nozzle pressure-vaccum cracking thalline repeatedly in the precipitation
1.3 add 100 μ l, 10% SDS;
1.4 add 100 μ g Proteinase Ks, incubation 1h in 50 ℃ of water-baths.
2. aqueous phase separation:
2.1 in every milliliter cracking digestion product, add the neutral saturated salt solution (NaCl) of 350 μ l;
2.2 above-mentioned solution is put upside down mixing 15s gently, at room temperature places 10min then;
2.3 the centrifugal 15min of room temperature 2500rpm, the supernatant aqueous phase then contains DNA.
3.DNA precipitation
3.1 shift the extremely new Eppendorf pipe of supernatant that contains DNA;
3.2 the centrifugal 10min of room temperature 12000rpm, supernatant discarded
1.3.DNA precipitation
4.DNA washing
4.1DNA precipitation adds 75% ethanol;
4.2 the centrifugal 5min of room temperature 10000rpm, supernatant discarded;
5.DNA dissolving
5.1 dry DNA precipitation 5min is dissolved in the ultrapure water then under the room temperature condition;
5.2 being carried out quantitatively also packing, the DNA that obtains is stored in-80 ℃.
6. remove the RNA pollutent
6.1 add the RNase of 50 μ g in every milliliter the dna solution;
6.2 37 ℃ of incubation 1h.
Annotate: removal RNA pollutes employed RNase facture can only be used in the final step that extracts.Desalination obtains high-quality DNA if desired, and RNase handles the step 3 and 4 of available the methodology in back and carries out.If will preserve DNA for a long time, available Tris-EDTA (TE) solution dissolving DNA is to suppress nuclease.
Two, RNA extracts operation scheme
1. homogenate
1.1 the bacterial cultures of 1ml is transferred to the centrifugal 5min acquisition of 2000rpm precipitation in the Eppendorf pipe
1.2 supernatant discarded adds the 1ml lysate and uses pipettor suction nozzle pressure-vaccum cracking thalline repeatedly in the precipitation
1.3 add the 10%SDS of 100 μ l;
1.4 add 100 μ g Proteinase Ks, incubation 1h in 50 ℃ of water-baths.
2. be separated
2.1 in every milliliter cracking digestion product, add 350 μ l pH and be 5 acid saturated salt solution (NaCl)
2.2 above-mentioned solution is put upside down mixing 15s gently, at room temperature places 10min then;
2.3 the centrifugal 15min of room temperature 2500rpm, the supernatant aqueous phase then contains RNA.
3.RNA precipitation
3.1 shift the extremely new Eppendorf pipe of supernatant that contains RNA;
3.2 the centrifugal 10min of room temperature 12000rpm, supernatant discarded;
4.RNA washing
4.1RNA precipitation adds 75% ethanol;
4.2 the centrifugal 5min of room temperature 10000rpm, supernatant discarded;
5.RNA dissolving
5.1 dry RNA precipitation 5min is dissolved in the ultrapure water after DEPC handles then under the room temperature condition;
5.2 being carried out quantitatively also packing, the RNA that obtains is stored in-80 ℃.
6. remove the genomic dna pollutent
The available DNase I of this part (no RNase) test kit is operated, and concrete steps are referring to the specification sheets of manufacturers.
Three, DNA separating effect:
The DNAA26o/A28o ratio that extracts is 1.83, and its quality meets follow-up molecular biology operational requirement fully.The output of DNA depends on the amount of initial organization material, promptly finally can obtain cultivating from the intestinal bacteria of 1ml incubated overnight obtaining 48 μ gDNA.Fig. 8 is the 1.5% agarose gel electrophoresis figure of the DNA of extraction.
Four, RNA extracts
The RNAA26o/A28o ratio that extracts is 1.99, and its quality meets follow-up molecular biology operational requirement fully.The output of RNA depends on the quantity of initial egg capsule, promptly 1 * 10
6Individual intestinal bacteria can obtain 22 μ gRNA.Fig. 9 is the 1.5% agarose gel electrophoresis figure of the RNA of extraction.
Five, the genomic dna of use the present invention extraction and RNA are in the application of downstream process
Available special primer is the specific target gene of pcr amplification from the DNA that extracts or RNA, DNA that extracts with checking or the applicability of RNA.Use in this example at colibacillary a plurality of 16S rDNA genes (gene accession number J01859/K02555/M24828/M24833/M24834/M24835/M24836/M24837/ M24911/M24996)) from the bacillus coli gene group DNA that is extracted, carry out the amplification of 16S rDNA.Employed primer is synthesized by the biological company limited of Beijing AudioCodes.The results are shown in Figure 10.
Claims (6)
1. a method of extracting DNA or RNA from protokaryon or eukaryote source material is characterized in that, comprises the steps:
1) pre-treatment: cracking protokaryon or eukaryote material also carry out dissolution process with stain remover, obtain the cracking digestion product;
2) saturated aqueous common salt is joined in the cracking digestion product, the albumen in the biomaterial is carried out precipitation process;
3) precipitation separation mutually and liquid phase, then to DNA in the liquid phase or RNA separates and purifying.
2. the method for claim 1 is characterized in that step 2) described salt solution is neutral or acid saturated brine, if need obtain DNA, then uses neutral saturated brine; If need obtain RNA, then use the acid saturated brine of pH value as 5-6.
3. method as claimed in claim 1 or 2 is characterized in that step 2) volumetric usage of described saturated aqueous common salt be cracking digestion product volume 0.3-1 doubly.
4. the method for claim 1 is characterized in that, in step 1), described cracking adopts the liquid nitrogen grinding method to carry out.
5. the method for claim 1 is characterized in that, in step 1), described cracking employing pipettor suction nozzle pressure-vaccum method repeatedly carries out.
6. the method for claim 1 is characterized in that, in step 1), described cracking adopts the granulated glass sphere method of breaking to carry out.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100092550A CN102140451A (en) | 2010-12-09 | 2011-01-17 | Method for extracting DNA (Desoxyribonucleic Acid) and RNA (Ribonucleic Acid) |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010593961.X | 2010-12-09 | ||
| CN201010593961 | 2010-12-09 | ||
| CN2011100092550A CN102140451A (en) | 2010-12-09 | 2011-01-17 | Method for extracting DNA (Desoxyribonucleic Acid) and RNA (Ribonucleic Acid) |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102140451A true CN102140451A (en) | 2011-08-03 |
Family
ID=44408248
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011100092550A Pending CN102140451A (en) | 2010-12-09 | 2011-01-17 | Method for extracting DNA (Desoxyribonucleic Acid) and RNA (Ribonucleic Acid) |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102140451A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286467A (en) * | 2011-08-30 | 2011-12-21 | 中国科学院亚热带农业生态研究所 | Method for extracting microbial total RNA in forest soil and litter |
| CN103421891A (en) * | 2013-03-26 | 2013-12-04 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Preparation methond of blood plasma miRNA used for PCR detection |
| CN107667168A (en) * | 2015-05-27 | 2018-02-06 | 凯杰有限公司 | Composition and method for disrupting tissue material |
| CN109402108A (en) * | 2017-08-18 | 2019-03-01 | 上海叶知生物医药科技有限公司 | The extracting method of RNA in a kind of extracellular vesica |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1635134A (en) * | 2004-10-22 | 2005-07-06 | 哈尔滨医科大学 | DNA extraction reagent and method for extracting mammal DNA by employing the reagent |
| CN1637012A (en) * | 2003-11-07 | 2005-07-13 | 株式会社日立高新技术 | RNA extraction method, RNA extraction reagent, and method for analyzing biological materials |
| CN101372689A (en) * | 2007-08-22 | 2009-02-25 | 中国科学院沈阳应用生态研究所 | Soil microbe genome DNA extracting method |
-
2011
- 2011-01-17 CN CN2011100092550A patent/CN102140451A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1637012A (en) * | 2003-11-07 | 2005-07-13 | 株式会社日立高新技术 | RNA extraction method, RNA extraction reagent, and method for analyzing biological materials |
| CN1635134A (en) * | 2004-10-22 | 2005-07-06 | 哈尔滨医科大学 | DNA extraction reagent and method for extracting mammal DNA by employing the reagent |
| CN101372689A (en) * | 2007-08-22 | 2009-02-25 | 中国科学院沈阳应用生态研究所 | Soil microbe genome DNA extracting method |
Non-Patent Citations (4)
| Title |
|---|
| MILLER ET AL: "A simple salting out procedure for extracting DNA from human nucleated cells", 《NUCLEIC ACIDS RESEARCH》 * |
| 刘开会，李宗亮: "《实用法医DNA检验学》", 31 December 2000 * |
| 涂向东等: "三种简易提取全血基因组DNA方法的比较", 《CHIN J LAB DIAGN》 * |
| 谭建明等: "快速盐析法提取DNA在HLA基因分型中的应用", 《中华器官移植杂志》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286467A (en) * | 2011-08-30 | 2011-12-21 | 中国科学院亚热带农业生态研究所 | Method for extracting microbial total RNA in forest soil and litter |
| CN102286467B (en) * | 2011-08-30 | 2012-11-28 | 中国科学院亚热带农业生态研究所 | Method for extracting microbial total RNA in forest soil and litter |
| CN103421891A (en) * | 2013-03-26 | 2013-12-04 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Preparation methond of blood plasma miRNA used for PCR detection |
| CN103421891B (en) * | 2013-03-26 | 2016-03-02 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | A kind of blood plasma miRNA preparation method detected for PCR |
| CN107667168A (en) * | 2015-05-27 | 2018-02-06 | 凯杰有限公司 | Composition and method for disrupting tissue material |
| CN109402108A (en) * | 2017-08-18 | 2019-03-01 | 上海叶知生物医药科技有限公司 | The extracting method of RNA in a kind of extracellular vesica |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20080160528A1 (en) | Use of Nucleases for Degrading Nucleic Acids in the Presence of Chaotropic Agents and/or Surfactants | |
| JP5112064B2 (en) | Kits and methods for removing contaminants from nucleic acids in environmental and biological samples | |
| CN101413018B (en) | Method for extracting genome DNA | |
| CN103097532B (en) | The method of target nucleic acid of the high yield separation including small target nucleic acid | |
| Nishiguchi et al. | DNA isolation procedures | |
| CN102242115B (en) | Kit for extracting bacterial plasmid DNA (deoxyribonucleic acid) by magnetic beads, and extraction method thereof | |
| US6503716B1 (en) | Compositions and methods for extracting a nucleic acid | |
| CN104603267B (en) | Separation includes the method for the RNA of tiny RNA with high yield | |
| CN102203251A (en) | Methods and kits for extraction of DNA | |
| CN104178480B (en) | Using the kit and method of DNA adsorption column rapid extraction DNA of plants | |
| CN102140451A (en) | Method for extracting DNA (Desoxyribonucleic Acid) and RNA (Ribonucleic Acid) | |
| US20210147915A1 (en) | Crispr/cas and transposase based amplification compositions, systems and methods | |
| CN101709298B (en) | Soil DNA extracting method for evaluating diversity of microbial community of plant root system | |
| CN101712953A (en) | DNA extracting method for evaluating community diversity of the intestinal microorganisms of animals | |
| Tripathy et al. | Exploring rapid and efficient protocol for isolation of fungal DNA | |
| Blood et al. | Handbook for | |
| JP2019519228A (en) | Compositions, systems and methods for the removal of detergents from aqueous solutions | |
| CN103205417A (en) | Method for quickly extracting high-purity plasmid DNA (Deoxyribonucleic Acid) | |
| CN101979541A (en) | Rapid covalently closed circular DNA purification kit | |
| CN107365766B (en) | Method for extracting RNA of mould spore by mechanical crushing method | |
| CN103966204A (en) | Method of extracting high-quality DNA (Deoxyribonucleic Acid) of fin product | |
| Adhikari et al. | A simplified high yielding miniprep genomic DNA extraction protocol for three chemotypically different plant species | |
| CN105505916A (en) | Method for extracting high-quality genome DNA from dry suriana maritime leaf and kit thereof | |
| CN104975001A (en) | Method for nondestructive extraction of rapana venosa genome DNA | |
| Moslem et al. | An efficient method for DNA extraction from Cladosporioid fungi |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20110803 |