CN101532011A - Kit for rapidly extracting and purifying DNA/RNA from same sample and method thereof - Google Patents
Kit for rapidly extracting and purifying DNA/RNA from same sample and method thereof Download PDFInfo
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- CN101532011A CN101532011A CN200810101602A CN200810101602A CN101532011A CN 101532011 A CN101532011 A CN 101532011A CN 200810101602 A CN200810101602 A CN 200810101602A CN 200810101602 A CN200810101602 A CN 200810101602A CN 101532011 A CN101532011 A CN 101532011A
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Abstract
The invention relates to the field of molecular biology, in particular to a kit for rapidly extracting and purifying DNA/RNA from the same sample and a method thereof. The kit comprises lysis solution, pretreatment solution, RNA binding solution, DNA wash solution I, RNA wash solution I and DNA/RNA wash solution II, DNA elutriant, a DNA binding column, an RNA binding column and a binding column. The method comprises the steps such as schizolysis, pretreatment, filtration, specific binding of DNA, DNA washing and purification, DNA eluting, specific binding of RNA, RNA washing and purification, RNA eluting and the like. The invention adopts novel media comprising a filtration medium and a binding medium, a new unique solution formula and combines the use of a specific inhibitor so as to greatly reduce the time of purification and greatly improve the purity and stability.
Description
Technical field
The present invention relates to biology field, relate in particular to a kind of test kit and the method for while extracting and purifying DNA/RNA from same sample fast.
Background technology
DNA and RNA are the base substances of heredity, are the carrier of genetic information, also are albumen synthetic templates, and the DNA of separating high-purity and RNA are modern molecular biology research and association area, comprise; The necessary technology means of agricultural, forestry, medical science, legal medical expert's evaluation and clinical diagnose, molecular therapy etc. also are impassable processs of the test.DNA and RNA have easy degraded, the characteristics of difficult purifying.It is easily by ubiquitous DNAse and RNase. degraded, also is easy in extractive process to be polluted each other by albumen and DNA and RNA; Traditional method, the separation and purification overwhelming majority of DNA and RNA is to carry out separately, that is: a same sample extracting RNA or DNA, as: alkaline lysis, the PEG precipitator method, AGPC method or acidic phenol method, cesium chloride density gradient centrifugation, Miniprep method etc.They all are that extracting is wherein a kind of: RNA or DNA.Trizol method commonly used was now once mentioned and can be obtained DNA simultaneously, but the purity extreme difference, big multi-method all must be used extremely strong phenol of toxicity and chloroform etc., and environment and testing crew are easily worked the mischief.Modern molecular medicine and molecular biology research all must detect DNA and RNA simultaneously, the present invention has abandoned independent extraction method in the past, created a kind of brand-new DNA and RNA extracting and purifying method simultaneously, while separation and purification DNA and RNA from same sample, to some preciousness, the sample that is difficult to obtain is particularly important, because it can save sample greatly, simultaneously also can shorten experimental period, this method is fit to purify from various materials and can be used for the high purity DNA and the RNA of the various experiments in downstream, as: PCR, gene chip, clone's enzyme is cut, Northernblot, cDNA transcribes, Northern blot, RNAi etc.
Summary of the invention
Therefore, in order to address the above problem, the present inventor proposes and has finished the present invention.
The present invention adopts novel medium, comprising: filtration medium and binding medium, and brand-new unique solution formula is used in combination special inhibitor, and the time of purification is significantly reduced, and purity, stability improves greatly.Its principle is as follows:
DNA and RNA fast purifying utilize strong denaturant (GuSCN etc.) and suitable buffering system smudge cells, suppress various Rnase, DNase, discharge DNA and RNA, utilize specific DNA/RNA adsorption medium and adsorbent solution, in conjunction with DNA/RNA, obtain the DNA and the RNA of purifying simultaneously through wash-out respectively.This invention will be the research and the application of bio-science and association area, provide DNA/RNA fast separating and purifying simultaneously, nuisanceless experimental technique; Also be the DNA/RNA separation and purification of extensive clinical diagnosis sample, comprise the separation and purification of viral DNA/RNA, provide simply, select effectively, efficiently.
Purpose of the present invention is for providing a kind of test kit of while extracting and purifying DNA/RNA from same sample fast.
Another object of the present invention is for providing a kind of method of while extracting and purifying DNA/RNA from same sample fast.
According to of the present invention fast from same sample simultaneously the test kit of extracting and purifying DNA/RNA comprise:
1) sample dissociation liquid,
The prescription of described lysate is:
3.5-5.5M?GuSCN
2-20mM?MgCl
2
10-50mM?MES-NaOH，pH6.3
The 10-30mM Trisodium Citrate
1-10mM?EDTA?pH8.0
0.005-0.3％?TritonX-100
0.05-0.2％NP-40
The 0.01-0.8%N-Sarkosyl L
The 1-8% propyl alcohol
The 5-20ul/ml beta-mercaptoethanol;
2) DNA column pre-treatment solution, its prescription is:
35-4.5M?LiCl
0.005-0.02M?HCl
0.05-0.3％?TritonX-100；
3) DNA washings I, its prescription is:
4.5-5.5M?GuHCl
5-20mM?MES，pH5.5
0.5-3mM?MgCl
2
0.02-0.06％?Triton?X-100
45-60% ethanol;
4) RNA binding soln, its prescription is;
250-750mM?MES-NaOH?pH5.0
65-80% ethanol;
5) RNA washing lotion I, its prescription is:
50-200mM?MES-NaOH，pH5～6，
3.5-4.5M?GuSCN
0.4-1％?TritonX-100
35-45% ethanol;
6) DNA/RNA washing lotion II,
Prescription for the described washing lotion II of vegetable material is:
70-85% ethanol
0.5-1.5% acetone
0.005-0.05％?DEPC
Prescription for the described washing lotion II of other material is:
70-85% ethanol and 0.005-0.05% DEPC;
7)Rnase-free?H
2O；
8) DNA elutriant, its prescription is:
5-15mM?Tris-HCl，pH8.0
1-2mM?EDTA
9) DNA column;
10) RNA column; And
11) Filter column.
According to test kit of the present invention, described DNA column is that the silica gel of 0.5-0.9 μ M is made by the aperture.
According to test kit of the present invention, the filtration medium of described Filter column is the aluminum oxide filter disc in 5-10 μ M aperture.
According to test kit of the present invention, described RNA column is that the glass fiber material of 0.3-0.8 μ M is made by the aperture.
According to of the present invention fast from same sample simultaneously the method for extracting and purifying DNA/RNA may further comprise the steps:
1) lysate sample is ground sample, and adds sample dissociation liquid and carry out cracking,
The prescription of described lysate is:
3.5-5.5M?GuSCN
2-20mM?MgCl
2
10-50mM?MES-NaOH，pH6.3
The 10-30mM Trisodium Citrate
1-10mM?EDTA?pH8.0
0.005-0.3％?TritonX-100
0.05-0.2％NP-40
The 0.01-0.8%N-Sarkosyl L
The 1-8% propyl alcohol
The 5-20ul/ml beta-mercaptoethanol;
2) pre-treatment is injected DNA column and RNA column respectively with 200 μ L pretreatment liquids, centrifugal then removal solution,
The prescription of described pre-treatment solution is:
3.5-4.5M?LiCl
0.005-0.02M?HCl
0.05-0.3％?TritonX-100；
3) filter, lysate sample is filtered with Filter column, collect filtrate;
4) specific combination DNA goes up sample to the DNA column with the filtrate that obtains in the step 3), and centrifugal collection filtrate for later use;
5) clean purification DNA, add 500 μ LDNA washing lotion I in the DNA column, and centrifugal removal solution, and then add 750 μ LDNA/RNA washing lotion II in the DNA column, and centrifugal removal solution,
The prescription of described DNA washings I is:
4.5-5.5M?GuHCl
5-20mM?MES，pH5.5
0.5-3mM?MgCl
2
0.02-0.06％?Triton?X-100
45-60% ethanol;
Described DNA/RNA washing lotion II,
Prescription for the described washing lotion II of vegetable material is:
70-85% ethanol
0.5-1.5% acetone
0.005-0.05％?DEPC
Prescription for the described washing lotion II of other material is:
70-85% ethanol and 0.005-0.05%DEPC
6) eluted dna adds 25-70 μ LDNA elutriant in the DNA column of handling through step 5), static, and centrifugal collection solution, obtains highly purified DNA,
The prescription of described DNA elutriant is:
5-15mM?Tris-HCl，pH8.0
1-2mM?EDTA
7) specific combination RNA adds isopyknic RNA binding soln and mixing in the filtrate that above-mentioned step 4) is collected, go up sample then to the RNA column, and centrifugal removal solution,
Described RNA in conjunction with the prescription of liquid is:
250-750mM?MES-NaOH?pH5.0
65-80% ethanol;
8) clean purification RNA, add 500 μ LRNA washing lotion I in the RNA column, and centrifugal removal solution, and then add 750 μ LRNA/DNA washing lotion II in the RNA column, and centrifugal removal solution,
The prescription of RNA washing lotion I is:
50-200mM?MES-NaOH，pH5～6，
3.5-4.5M?GuSCN
0.4-1％?TitonX-100
35-45% ethanol;
9) eluted rna, the H2O of adding Rnase-free is static in the RNA column of handling through step 8), and centrifugal collection solution, obtains highly purified RNA.
The method according to this invention, described DNA column are that the silica gel of 0.5-0.9 μ M is made by the aperture.
The method according to this invention, the filtration medium of described Filter column are the aluminum oxide filter disc in 5-10 μ M aperture.
The method according to this invention, described RNA column are that the glass fiber material of 0.3-0.8 μ M is made by the aperture.
To sum up, the present invention has the following advantages:
1, abandon in the past that RNA and dna single solely extract, waste sample and the conventional classical way of time are used same sample, extract RNA and DNA simultaneously.
2, adopt gentle denaturing agent cracking process, commercialization different from the past " Trizol " method, cesium chloride density gradient centrifugation and the used phenol extraction process of acidic phenol method use the medicine that environment is polluted.
3, sample is collected in not centrifugation, a direct cracking of step, and two go on foot combination respectively, special cleaning purifying.
4, adopt special brand-new solution formula pre-treatment silicon dioxide fibrous membrane, specific combination DNA or RNA do not pollute albumen, DNA and RNA mutually, and other impurity are easy-clear more.
5, the separation purification time is taken out in shortening greatly, can finish in 15 minutes.
6, this method is that DNA and RNA extractive automatization simultaneously and extensiveization have been established technical foundation.
Embodiment
Embodiment one uses total RNA/DNA of the quick extracting and purifying tobacco leaf of test kit of the present invention
One, obtain solution
Prepare following solution:
1) sample dissociation liquid,
4M?GuSCN
2.5mM?MgCl
2
25mM?MES-NaOH，pH6.3
The 20mM Trisodium Citrate
2mM?EDTApH8.0
0.1％?TritonX-100
0.15％NP-40
The 0.5%N-Sarkosyl L
5% propyl alcohol
The 10ul/ml beta-mercaptoethanol;
2) DNA column pre-treatment solution:
4M?LiCl
0.01M?HCl
0.1％TritonX-100；
3) DNA washings I:
4.5M?GuHCl
10mM?MES，pH5.5
1mM?MgCl
2
0.05％Triton?X-100
55% ethanol;
4) RNA binding soln:
500mM?MES-NaOH?pH5.0
70% ethanol;
5) RNA washing lotion I:
100mM?MES-NaOH，pH5～6，
4M?GuSCN
0.7％?TritonX-100
40% ethanol;
6) DNA/RNA washing lotion II,
Prescription for the described washing lotion II of vegetable material is:
78% ethanol
1% acetone
0.01％DEPC，
Prescription for the described washing lotion II of other material is:
78% ethanol and 0.01%DEPC;
7)Rnase-free?H
2O；
8) DNA elutriant:
10mM?Tris-HCl，pH8.0
1mM?EDTA。
Two, extract total RNA and the DNA of tobacco
1, lysate sample
Sample thief (being about 50mg) is put into liquid nitrogen and is moved in turn and grind, and adds lysate 350 μ l, concuss, and 60 ℃ of incubations left standstill 5 minutes.
2, while pre-treatment DNA and RNA column: 200 μ L DNA pre-treatment solution are added in the post centrifugal 10 seconds of 11000rpm, reject solution.
3, filter, lysate sample is filtered with the wide aperture Filter column, collect filtrate;
4, specific combination DNA goes up sample to the DNA column with the filtrate that obtains in the step 3, and 11,000rpm (8200 * g) centrifugal 20 seconds collection filtrate for later use;
5, clean purification DNA
Add 500 μ l DNA washing lotion I in the DNA column, 11,000rpm (8200 * g) centrifugal 20 seconds, reject solution.Add 750 μ lRNA/DNA washing lotion II again, 11,000rpm (8200 * g) centrifugal 10 seconds, reject solution, centrifugal again 1 minute.
6, wash-out high purity DNA
In conjunction with the dry post of DNA, add 53 μ l DNA elutriants with, left standstill 1 minute, 11,000rpm (8200 * g) centrifugal 1 minute, collect solution, high purity DNA be about 50 μ L (0.4 μ g/ μ L).
7, specific combination RNA, the filtrate that step 4 is collected adds isopyknic RNA binding soln, mixing, all samples (filtrate) is gone up sample to the RNA column, and 11, centrifugal 20 seconds of 000rpm (8200xg).
8, clean purification RNA
Add 500 μ l RNA dilution I in the RNA column, 11, centrifugal 20 seconds of 000rpm (8200xg), reject solution.Add 750 μ l RNA/DNA washing soln II again, 11, centrifugal 10 seconds of 000rpm (8200xg), reject solution, centrifugal again 1 minute.
9, wash-out high purity RNA
In conjunction with the dry post of RNA, add the RNA elutriant with, left standstill 1 minute, 11, centrifugal 1 minute of 000rpm (8200xg) collects solution, gets high purity RNA and is about 50 μ l (0.4 μ g/ μ l).
Embodiment two extracts the total RNA and the DNA of yeast cell
With the method identical, extract the total RNA and the DNA of yeast cell with embodiment one.
Embodiment three uses total RNA/DNA of the quick extracting and purifying tobacco leaf of test kit of the present invention
One, obtain solution
Prepare following solution:
1) sample dissociation liquid,
3.5M?GuSCN
2mM?MgCl
2
10mM?MES-NaOH，pH6.3
The 10mM Trisodium Citrate
1mM?EDTA?pH8.0
0.005％?TritonX-100
0.05％NP-40
The 0.01%N-Sarkosyl L
1% propyl alcohol
The 5ul/ml beta-mercaptoethanol;
2) DNA column pre-treatment solution:
3.5M?LiCl
0.005M?HCl
0.05％?TritonX-100；
3) DNA washings I:
4.5M?GuHCl
5mM?MES，pH5.5
0.5mM?MgCl
2
0.02％?Triton?X-100
45% ethanol;
4) RNA binding soln:
50mM?MES-NaOH?pH5.0
65% ethanol;
5) RNA washing lotion I:
50mM?MES-NaOH，pH5～6，
3.5M?GuSCN
0.4％?TritonX-100
35% ethanol;
6) DNA/RNA washing lotion II,
Prescription for the described washing lotion II of vegetable material is:
70% ethanol
0.5% acetone
0.005％?DEPC，
Prescription for the described washing lotion II of other material is:
70% ethanol and 0.005%DEPC;
7)Rnase-free?H
2O；
8) DNA elutriant:
5mM?Tris-HCl，pH8.0
1mM?EDTA。
Two, extract total RNA and the DNA of tobacco
1, lysate sample
Sample thief (being about 50mg) is put into liquid nitrogen and is moved in turn and grind, and adds lysate 350 μ l, concuss, and 60 ° of C of incubation left standstill 5 minutes.
2, while pre-treatment DNA and RNA column: 200 μ L DNA pre-treatment solution are added in the post centrifugal 10 seconds of 11000rpm, reject solution.
3, filter, lysate sample is filtered with the wide aperture Filter column, collect filtrate;
4, specific combination DNA goes up sample to the DNA column with the filtrate that obtains in the step 3, and 11,000rpm (8200 * g) centrifugal 20 seconds collection filtrate for later use;
5, clean purification DNA
Add 500 μ l DNA washing lotion I in the DNA column, 11,000rpm (8200 * g) centrifugal 20 seconds, reject solution.Add 750 μ lRNA/DNA washing lotion II again, 11,000rpm (8200 * g) centrifugal 10 seconds, reject solution, centrifugal again 1 minute.
6, wash-out high purity DNA
In conjunction with the dry post of DNA, add 53 μ l DNA elutriants with, left standstill 1 minute, 11,000rpm (8200 * g) centrifugal 1 minute, collect solution, high purity DNA be about 50 μ L (0.4 μ g/ μ L).
7, specific combination RNA, the filtrate that step 4 is collected adds isopyknic RNA binding soln, mixing, all samples (filtrate) is gone up sample to the RNA column, and 11, centrifugal 20 seconds of 000rpm (8200xg).
8, clean purification RNA
Add 500 μ l RNA dilution I in the RNA column, 11, centrifugal 20 seconds of 000rpm (8200xg), reject solution.Add 750 μ l RNA/DNA washing soln II again, 11, centrifugal 10 seconds of 000rpm (8200xg), reject solution, centrifugal again 1 minute.
9, wash-out high purity RNA
In conjunction with the dry post of RNA, add the RNA elutriant with, left standstill 1 minute, 11, centrifugal 1 minute of 000rpm (8200xg) collects solution, gets high purity RNA and is about 50 μ l (0.4 μ g/ μ l).
Embodiment four uses total RNA/DNA of the quick extracting and purifying tobacco leaf of test kit of the present invention
One, obtain solution
Prepare following solution:
1) sample dissociation liquid,
5.5M?GuSCN
20mM?MgCl
2
50mM?MES-NaOH，pH6.3
The 30mM Trisodium Citrate
10mM?EDTApH8.0
0.3％?TritonX-100
0.2％NP-40
The 0.8%N-Sarkosyl L
8% propyl alcohol
The 20ul/ml beta-mercaptoethanol;
2) DNA column pre-treatment solution:
4.5M?LiCl
0.02M?HCl
0.3％?TritonX-100；
3) DNA washings I:
5.5M?GuHCl
20mM?MES，pH5.5
3mM?MgCl
2
0.06％?Triton?X-100
60% ethanol;
4) RNA binding soln:
750mM?MES-NaOH?pH5.0
80% ethanol;
5) RNA washing lotion I:
200mM?MES-NaOH，pH5～6，
4.5M?GuSCN
1％?TritonX-100
45% ethanol;
6) DNA/RNA washing lotion II,
Prescription for the described washing lotion II of vegetable material is:
85% ethanol
1.5% acetone
0.05％?DEPC，
Prescription for the described washing lotion II of other material is:
85% ethanol and 0.05%DEPC;
7)Rnase-free?H
2O；
8) DNA elutriant:
15mM?Tris-HCl，pH8.0
2mM?EDTA。
Two, extract total RNA and the DNA of tobacco
1, lysate sample
Sample thief (being about 50mg) is put into liquid nitrogen and is moved in turn and grind, and adds lysate 350 μ l, concuss, and 60 ℃ of incubations left standstill 5 minutes.
2, while pre-treatment DNA and RNA column: 200 μ L DNA pre-treatment solution are added in the post centrifugal 10 seconds of 11000rpm, reject solution.
3, filter, lysate sample is filtered with the wide aperture Filter column, collect filtrate;
4, specific combination DNA goes up sample to the DNA column with the filtrate that obtains in the step 3, and 11,000rpm (8200 * g) centrifugal 20 seconds collection filtrate for later use;
5, clean purification DNA
Add 500 μ l DNA washing lotion I in the DNA column, 11,000rpm (8200 * g) centrifugal 20 seconds, reject solution.Add 750 μ lRNA/DNA washing lotion II again, 11,000rpm (8200 * g) centrifugal 10 seconds, reject solution, centrifugal again 1 minute.
6, wash-out high purity DNA
In conjunction with the dry post of DNA, add 53 μ l DNA elutriants with, left standstill 1 minute, 11,000rpm (8200 * g) centrifugal 1 minute, collect solution, high purity DNA be about 50 μ L (0.4 μ g/ μ L).
7, specific combination RNA, the filtrate that step 4 is collected adds isopyknic RNA binding soln, mixing, all samples (filtrate) is gone up sample to the RNA column, and 11, centrifugal 20 seconds of 000rpm (8200xg).
8, clean purification RNA
Add 500 μ l RNA dilution I in the RNA column, 11, centrifugal 20 seconds of 000rpm (8200xg), reject solution.Add 750 μ l RNA/DNA washing soln II again, 11, centrifugal 10 seconds of 000rpm (8200xg), reject solution, centrifugal again 1 minute.
9, wash-out high purity RNA
In conjunction with the dry post of RNA, add the RNA elutriant with, left standstill 1 minute, 11, centrifugal 1 minute of 000rpm (8200xg) collects solution, gets high purity RNA and is about 50 μ l (0.4 μ g/ μ l).
Claims (8)
1, a kind of test kit of while extracting and purifying DNA/RNA from same sample fast is characterized in that described test kit comprises following component:
1) sample dissociation liquid,
The prescription of described lysate is:
3.5-5.5M?GuSCN
2-20mM?MgCl
2
10-50mM?MES-NaOH，pH6.3
The 10-30mM Trisodium Citrate
1-10mM?EDTA?pH8.0
0.005-0.3％?TritonX-100
0.05-0.2％NP-40
0.01-0.8% N-Sarkosyl L
The 1-8% propyl alcohol
The 5-20ul/ml beta-mercaptoethanol;
2) column pre-treatment solution, its prescription is:
3.5-4.5M?LiCl
0.005-0.02M?HCl
0.05-0.3％?TritonX-100；
3) DNA washings I, its prescription is:
4.5-5.5M?GuHCl
5-20mM?MES，pH5.5
0.5-3mM?MgCl
2
0.02-0.06％?Triton?X-100
45-60% ethanol;
4) RNA binding soln, its prescription is;
250-750mM?MES-NaOH?pH5.0
65-80% ethanol;
5) RNA washing lotion I, its prescription is:
50-200mM?MES-NaOH，pH5～6，
3.5-4.5M?GuSCN
0.4-1％?TritonX-100
35-45% ethanol;
6) DNA/RNA washing lotion II,
Prescription for the described washing lotion II of vegetable material is:
70-85% ethanol
0.5-1.5% acetone
0.005-0.05％?DEPC
Prescription for the described washing lotion II of other material is:
70-85% ethanol and 0.005-0.05% DEPC;
7)Rnase-free?H
2O；
8) DNA elutriant, its prescription is:
5-15mM?Tris-HCl，pH8.0
1-2mM?EDTA；
9) DNA column;
10) RNA column; And
11) Filter column.
2, test kit according to claim 1 is characterized in that, described DNA column is that the silica gel of 0.5-0.9 μ M is made by the aperture.
3, test kit according to claim 1 is characterized in that, the filtration medium of described Filter column is the aluminum oxide filter disc in 5-10 μ M aperture.
4, test kit according to claim 1 is characterized in that, described RNA column is that the glass fiber material of 0.3-0.8 μ M is made by the aperture.
5, a kind of method of while extracting and purifying DNA/RNA from same sample fast is characterized in that, said method comprising the steps of:
1) lysate sample is ground sample, and adds sample dissociation liquid and carry out cracking,
The prescription of described lysate is:
3.5-5.5M?GuSCN
2-20mM?MgCl
2
10-50mM?MES-NaOH，pH6.3
The 10-30mM Trisodium Citrate
1-10mM?EDTA?pH8.0
0.005-0.3％?Triton?X-100
0.05-0.2％NP-40
0.01-0.8% N-Sarkosyl L
The 1-8% propyl alcohol
The 5-20ul/ml beta-mercaptoethanol;
2) pre-treatment is injected DNA column and RNA column respectively with 200 μ L pretreatment liquids, centrifugal then removal solution,
The prescription of described pre-treatment solution is:
3.5-4.5M?LiCl
0.005-0.02M?HCl
0.05-0.3％?Triton?X-100；
3) filter, lysate sample is filtered with Filter column, collect filtrate;
4) specific combination DNA goes up sample to the DNA column with the filtrate that obtains in the step 3), and centrifugal collection filtrate for later use;
5) clean purification DNA, add 500 μ LDNA washing lotion I in the DNA column, and centrifugal removal solution, and then add 750 μ L DNA/RNA washing lotion II in the DNA column, and centrifugal removal solution,
The prescription of described DNA washings I is:
4.5-5.5M?GuHCl
5-20mM?MES，pH5.5
0.5-3mM?MgCl
2
0.02-0.06％?Triton?X-100
45-60% ethanol;
Described DNA/RNA washing lotion II,
Prescription for the described washing lotion II of vegetable material is:
70-85% ethanol
0.5-1.5% acetone
0.005-0.05％?DEPC
Prescription for the described washing lotion II of other material is:
70-85% ethanol and 0.005-0.05% DEPC;
6) eluted dna adds 53 μ L DNA elutriants in the DNA column of handling through step 5), static, and centrifugal collection solution, obtains highly purified DNA,
The prescription of described DNA elutriant is:
5-15mM?Tris-HCl，pH8.0
1-2mM?EDTA；
7) specific combination RNA adds isopyknic RNA binding soln and mixing in the filtrate that above-mentioned step 4) is collected, go up sample then to the RNA column, and centrifugal removal solution,
Described RNA in conjunction with the prescription of liquid is:
250-750mM?MES-NaOH?pH5.0
65-80% ethanol;
8) clean purification RNA, add 500 μ L RNA washing lotion I in the RNA column, and centrifugal removal solution, and then add 750 μ L RNA/DNA washing lotion II in the RNA column, and centrifugal removal solution,
The prescription of RNA washing lotion I is:
50-200mM?MES-NaOH，pH5～6，
3.5-4.5M?GuSCN
0.4-1％?Triton?X-100
35-45% ethanol;
9) eluted rna, the H of adding Rnase-free in the RNA column of handling through step 8)
2O, static, and centrifugal collection solution, obtain highly purified RNA.
6, method according to claim 5 is characterized in that, described DNA column is that the silica gel of 0.5-0.9 μ M is made by the aperture.
7, method according to claim 5 is characterized in that, the filtration medium of described Filter column is the aluminum oxide filter disc in 5-10 μ M aperture.
8, method according to claim 5 is characterized in that, described RNA column is that the glass fiber material of 0.3-0.8 μ M is made by the aperture.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101016020A CN101532011B (en) | 2008-03-10 | 2008-03-10 | Kit for rapidly extracting and purifying DNA/RNA from same sample and method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101016020A CN101532011B (en) | 2008-03-10 | 2008-03-10 | Kit for rapidly extracting and purifying DNA/RNA from same sample and method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101532011A true CN101532011A (en) | 2009-09-16 |
| CN101532011B CN101532011B (en) | 2010-12-08 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN2008101016020A Expired - Fee Related CN101532011B (en) | 2008-03-10 | 2008-03-10 | Kit for rapidly extracting and purifying DNA/RNA from same sample and method thereof |
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| CN101948828A (en) * | 2010-10-14 | 2011-01-19 | 江苏省农业科学院 | Trace animal cell genomic DNA template preparation solution and corresponding DNA template preparation method |
| US8202693B2 (en) * | 2009-11-24 | 2012-06-19 | Omega Bio-Tek, Inc. | Method of isolation of nucleic acids |
| CN112501158A (en) * | 2020-12-04 | 2021-03-16 | 麦凯(上海)生物科技有限公司 | Human liquid sample whole nucleic acid extraction kit and application thereof |
| CN113493783A (en) * | 2021-07-23 | 2021-10-12 | 杭州圣庭医疗科技有限公司 | Method for co-extracting DNA and RNA of different samples |
| CN114426965A (en) * | 2020-10-29 | 2022-05-03 | 深圳华大生命科学研究院 | Kit for simultaneously extracting DNA, RNA and protein from plant tissue sample and general method |
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| CN1086201C (en) * | 1999-01-29 | 2002-06-12 | 朱明华 | Reagent box for extracting DNA fast and use thereof |
| CN1313478C (en) * | 2004-12-10 | 2007-05-02 | 四川农业大学 | New method for rapid purifying DNA plasmid in large quantities |
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| US8202693B2 (en) * | 2009-11-24 | 2012-06-19 | Omega Bio-Tek, Inc. | Method of isolation of nucleic acids |
| CN101948828A (en) * | 2010-10-14 | 2011-01-19 | 江苏省农业科学院 | Trace animal cell genomic DNA template preparation solution and corresponding DNA template preparation method |
| CN101948828B (en) * | 2010-10-14 | 2011-08-17 | 江苏省农业科学院 | Trace animal cell genomic DNA template preparation solution and corresponding DNA template preparation method |
| CN114426965A (en) * | 2020-10-29 | 2022-05-03 | 深圳华大生命科学研究院 | Kit for simultaneously extracting DNA, RNA and protein from plant tissue sample and general method |
| CN114426965B (en) * | 2020-10-29 | 2023-12-26 | 深圳华大生命科学研究院 | Kit for simultaneously extracting DNA, RNA and protein from plant tissue sample and general method |
| CN112501158A (en) * | 2020-12-04 | 2021-03-16 | 麦凯(上海)生物科技有限公司 | Human liquid sample whole nucleic acid extraction kit and application thereof |
| CN113493783A (en) * | 2021-07-23 | 2021-10-12 | 杭州圣庭医疗科技有限公司 | Method for co-extracting DNA and RNA of different samples |
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| CN101532011B (en) | 2010-12-08 |
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