CN108531546A - The method of efficiently convenient extraction paddy DNA - Google Patents
The method of efficiently convenient extraction paddy DNA Download PDFInfo
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- CN108531546A CN108531546A CN201711498735.1A CN201711498735A CN108531546A CN 108531546 A CN108531546 A CN 108531546A CN 201711498735 A CN201711498735 A CN 201711498735A CN 108531546 A CN108531546 A CN 108531546A
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- CN
- China
- Prior art keywords
- pcr
- efficiently convenient
- convenient extraction
- paddy dna
- dna according
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Abstract
This application discloses a kind of methods of efficiently convenient extraction paddy DNA, include the following steps：(1) 200 holes μ L96 PCR plates are placed on ice, 2 fresh blades is taken to be put into PCR plate respectively with card punch；(2) 70 μ L buffer solution As are added per hole, are sealed with sealing plate film, tablet is put into PCR instrument is heated to 95 DEG C at once, keep 10min；(3) tablet is taken out, isometric buffer solution B, mixing are rapidly added.1 μ L of extracting solution in step (3) can carry out PCR detections directly as template DNA.The method of the present invention utilizes the efficiently convenient extraction paddy DNA of PCR plate, and shortens PCR loading times.
Description
Technical field
The present invention relates to molecular biology Biotechnology in Genetic Breeding field, relate in particular to it is a kind of using PCR plate efficiently just
The method of victory extraction paddy DNA.
Background technology
The extraction of DNA is the premise of plant molecular genetics and genetic engineering.Usual DNA extraction method needs to meet following
Several essential conditions：Gained DNA has ideal purity；DNA is complete, and fracture is few, and palliating degradation degree is small；It is convenient that method facilitates, at
This is low.It is to be engaged in DNA molecular marker worker's questions of common interest to improve the efficiency of genomic DNA preparation and reduce cost.
Currently, have many reports about oryza sativa genomic dna extracting method, but general extraction methods such as CTAB methods and a large amount of
The DNA extraction kit to appear on the market, although the DNA of high quality can be mentioned, extraction process is loaded down with trivial details, and cost is higher, is not easy to apply
In large-scale genotype detection operation.
And some are easily suitable for the needs such as molecular marker assisted selection, mapping population, mutant screening, Seed Inspection
Batch samples carry out the DNA extraction method of genotyping, although have it is many simplify, that there are still operating procedures is complicated,
The problems such as cost is higher or its amplification of template DNA prepared is not sufficiently stable or is only applicable to the extraction of leaf DNA, thus
It also needs further to explore efficient, quick, easy, low cost DNA extraction method
Invention content
Technical problem to be solved by the present invention lies in, a kind of utilization efficiently convenient extraction paddy DNA of PCR plate is provided,
And shorten the method for PCR loading times.
In order to solve the above technical problems, the present invention provides a kind of method of efficiently convenient extraction paddy DNA, including it is as follows
Step：
(1) 200 holes μ L96 PCR plates are placed on ice, 2 fresh blades is taken to be put into PCR plate respectively with card punch；
(2) 70 μ L buffer solution As are added per hole, is sealed with sealing plate film, tablet is put into PCR instrument is heated to 95 DEG C at once,
Keep 10min；
(3) tablet is taken out, isometric buffer solution B, mixing are rapidly added.
1 μ L of extracting solution in step (3) can carry out PCR detections directly as template DNA.
Further, PCR detections are carried out if you need to retain one week or more time, then draws 40 μ L of said extracted liquid with the volley of rifle fire
In 200 μ L, the 96 hole PCR orifice plates new to one piece, the TE buffer solutions of 3 times of extracting liquid volumes are added；4 DEG C of refrigerators are put into preserve, it is standby
With.
Buffer solution A described in step (2) is fresh solution.
Buffer solution A further comprises described in step (2)：800 μ L 20%Tween20.
Buffer solution A further comprises described in step (2)：160μL 5mol/L NaOH.
Buffer solution A further comprises described in step (2)：7.04mL ddH2O
Buffer solution B further comprises described in step (3)：1mL pH8.01mol/L Tris-HCl.
Buffer solution B further comprises described in step (3)：40μL pH8.00.5mol/LEDTA.
Buffer solution B further comprises described in step (3)：8.96mL ddH2O。
The beneficial technique effect of the present invention includes：
(1) present invention only uses 4 kinds of chemical reagent of NaOH, Tween20, Tris-HCl and EDTA, totally 140 μ L extracting solutions, at
This is low.
(2) present invention is easy to operate, it is only necessary to which 3 steps can extract thousands of parts of samples for each person every day.
(3) instrument and equipment of the present invention is simple, only needs conventional PCR instrument.
(4) present invention extracts DNA using 96 hole PCR plates, and extracting solution is pipetted using the volley of rifle fire, can shorten further PCR sample-addings
Time.
Description of the drawings
Fig. 1 is that salt is rich 47, long grain is fragrant, the PCR plate after excellent No. thousand super, wide Hunan 145 blade sampling；
Fig. 2 is rich 47 using salt, long grain is fragrant, excellent No. thousand super, wide Hunan 145 blade using PCR plate extraction DNA and tradition
(1-8 is the DNA electrophoretograms of PCR plate extraction to the comparison diagram of the DNA electrophoresis of CTAB methods extraction, and 9-16 is the extraction of traditional CT AB methods
Electrophoretogram)；
Fig. 3 is rich 47 using salt, long grain is fragrant, excellent No. thousand super, wide Hunan 145 dry seed using PCR plate extraction DNA and biography
(1-8 is the DNA electrophoretograms of PCR plate extraction to the comparison diagram of the DNA electrophoresis of CTAB methods of uniting extraction, and 9-16 extracts for traditional CT AB methods
Electrophoretogram)；
Fig. 4 is rich 47 using salt, long grain is fragrant, excellent No. thousand super, wide Hunan 145 root using PCR plate extraction DNA and tradition
(1-8 is the DNA electrophoretograms of PCR plate extraction to the comparison diagram of the DNA electrophoresis of CTAB methods extraction, and 9-16 is the extraction of traditional CT AB methods
Electrophoretogram).
Specific implementation mode
The present invention is described in detail with reference to embodiment.To keep the objectives, technical solutions, and advantages of the present invention clearer, bright
Really, developing simultaneously referring to the drawings, the present invention is described in more detail for embodiment, but the invention is not limited in these embodiments.
In order to solve the above-mentioned technical problem, the present invention adopts the following technical scheme that：It is a kind of to utilize the efficiently convenient extraction of PCR plate
The method of paddy DNA.
A method of using the efficient convenient extraction paddy DNA of PCR plate, include the following steps：
(1) 200 holes μ L96 PCR plates are placed on ice, 2 fresh blades is taken to be put into PCR plate (figure respectively with card punch
1)；
(2) 70 μ L buffer solution As are added per hole, is sealed with sealing plate film, tablet is put into PCR instrument is heated to 95 DEG C at once,
Keep 10min；
(3) tablet is taken out, isometric buffer solution B, mixing are rapidly added.
Further, the 1 μ L of extracting solution in step (3) can carry out PCR detections directly as template DNA
Further, PCR detections (after 1 week) are carried out if you need to retain a period of time, 40 μ of said extracted liquid is drawn with the volley of rifle fire
In 200 μ L, 96 hole PCR orifice plates new L (determining uptake as needed) to one piece, the TE bufferings of 3 times of extracting liquid volumes are added
Liquid.4 DEG C of refrigerators are put into preserve, it is spare.
Further, buffer solution A described in step (2) is fresh solution.
Further, buffer solution A described in step (2) includes：800 μ L 20%Tween20,160 μ L 5mol/L
NaOH, 7.04mL ddH2O (in terms of 96 hole PCR plate of monoblock)
Further, buffer solution B described in step (3) includes：1mL 1mol/L Tris-HCl (pH8.0), 40 μ L
0.5mol/LEDTA (pH8.0), 8.96mL ddH2O.
The present disclosure applies equally to extract the genomic DNA of LIPIDS OF DRY RICE EMBRYO seed or rice root.
Described in LIPIDS OF DRY RICE EMBRYO seed do not need decladding, but need to crush.
Described in LIPIDS OF DRY RICE EMBRYO seed the half of no embryo is cut with blade if taking half seed extraction DNA.
The present invention also provides the application of the method for above-mentioned DNA extractions in rice.
Embodiment 1
Material for extracting paddy DNA is：Salt is rich 47, long grain is fragrant, excellent No. thousand super, wide Hunan 145 blade.
200 holes μ L96 PCR plates are placed on ice, 2 fresh blades is taken to be put into PCR plate respectively with card punch.Every part of material
Material places two rows totally 24 hole.70 μ L buffer solution As are added per hole, is sealed with sealing plate film, tablet is put into PCR instrument is heated at once
95 DEG C, keep 10min；Tablet is taken out, isometric buffer solution B, mixing are rapidly added.
It takes 0.15g agar Icing Sugar, 15mL distilled water is added, be heated to micro-wave oven transparent, prepare 1% agarose gel.
Take the 10 μ L of extracting solution in A1, B2, C3, D4, E5, F6, H7, I8, mixing Buffer that agarose gel hole is added respectively
In, 110V after 30min electrophoresis, is placed in gel imager and shoots.
Embodiment 2
Material for extracting paddy DNA is：Salt is rich 47, long grain is fragrant, excellent No. thousand super, wide Hunan 145 dry seed.
200 holes μ L96 PCR plates are placed on ice, after dry seed is crushed, are put into PCR plate.Every part of material places two rows
Totally 24 hole.70 μ L buffer solution As are added per hole, are sealed with sealing plate film, tablet is put into PCR instrument is heated to 95 DEG C at once, keep
10min；Tablet is taken out, isometric buffer solution B, mixing are rapidly added.
It takes 0.15g agar Icing Sugar, 15mL distilled water is added, be heated to micro-wave oven transparent, prepare 1% agarose gel.
Take the 10 μ L of extracting solution in A1, B2, C3, D4, E5, F6, H7, I8, mixing Buffer that agarose gel hole is added respectively
In, 110V after 30min electrophoresis, is placed in gel imager and shoots.
Embodiment 3
Material for extracting paddy DNA is：Salt is rich 47, long grain is fragrant, excellent No. thousand super, wide Hunan 145 root.
200 holes μ L96 PCR plates are placed on ice, take 10mg rice roots to be put into PCR plate (Fig. 1) with scissors；Every part of material
Place two rows totally 24 hole.70 μ L buffer solution As are added per hole, is sealed with sealing plate film, tablet is put into PCR instrument is heated to 95 at once
DEG C, keep 10min；Tablet is taken out, isometric buffer solution B, mixing are rapidly added.
It takes 0.15g agar Icing Sugar, 15mL distilled water is added, be heated to micro-wave oven transparent, prepare 1% agarose gel.
Take the 10 μ L of extracting solution in A1, B2, C3, D4, E5, F6, H7, I8, mixing Buffer that agarose gel hole is added respectively
In, 110V after 30min electrophoresis, is placed in gel imager and shoots.
The above is only several embodiments of the present invention, not any type of limitation is done to the present invention, although this hair
It is bright to be disclosed as above with preferred embodiment, however not to limit the present invention, any person skilled in the art is not taking off
In the range of technical solution of the present invention, makes a little variation using the technology contents of the disclosure above or modification is equal to
Case study on implementation is imitated, is belonged in technical solution of the present invention protection domain.
Claims (10)
1. a kind of method of efficiently convenient extraction paddy DNA, which is characterized in that include the following steps：
(1) 200 holes μ L96 PCR plates are placed on ice, 2 fresh blades is taken to be put into PCR plate respectively with card punch；
(2) 70 μ L buffer solution As are added per hole, are sealed with sealing plate film, tablet is put into PCR instrument is heated to 95 DEG C at once, keep
10min；
(3) tablet is taken out, isometric buffer solution B, mixing are rapidly added.
2. the method for efficiently convenient extraction paddy DNA according to claim 1, which is characterized in that further, step (3)
In 1 μ L of extracting solution can directly as template DNA carry out PCR detections.
3. the method for efficiently convenient extraction paddy DNA according to claim 1, which is characterized in that it is further, if you need to retain
One week or more time carried out PCR detections, then drew μ L to the one pieces of new 96 hole hole PCR 200 μ L of said extracted liquid 40 with the volley of rifle fire
In plate, the TE buffer solutions of 3 times of extracting liquid volumes are added；4 DEG C of refrigerators are put into preserve, it is spare.
4. the method for efficiently convenient extraction paddy DNA according to claim 1, which is characterized in that further, step (2)
Described in buffer solution A be fresh solution.
5. the method for efficiently convenient extraction paddy DNA according to claim 1, which is characterized in that delay described in step (2)
Fliud flushing A further comprises：800 μ L 20%Tween20.
6. the method for efficiently convenient extraction paddy DNA according to claim 1, which is characterized in that step buffers described in (2)
Liquid A further comprises：160μL 5mol/L NaOH.
7. the method for efficiently convenient extraction paddy DNA according to claim 1, which is characterized in that step buffers described in (2)
Liquid A further comprises：7.04mL ddH2O 。
8. the method for efficiently convenient extraction paddy DNA according to claim 1, which is characterized in that step buffers described in (3)
Liquid B further comprises：1mL pH8.01mol/L Tris-HCl.
9. the method for efficiently convenient extraction paddy DNA according to claim 1, which is characterized in that step buffers described in (3)
Liquid B further comprises：40μL pH8.00.5mol/LEDTA.
10. the method for efficiently convenient extraction paddy DNA according to claim 1, which is characterized in that delay described in step (3)
Fliud flushing B further comprises：8.96mL ddH2O。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711498735.1A CN108531546A (en) | 2017-12-30 | 2017-12-30 | The method of efficiently convenient extraction paddy DNA |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711498735.1A CN108531546A (en) | 2017-12-30 | 2017-12-30 | The method of efficiently convenient extraction paddy DNA |
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| Publication Number | Publication Date |
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Family
ID=63489820
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| CN201711498735.1A Withdrawn CN108531546A (en) | 2017-12-30 | 2017-12-30 | The method of efficiently convenient extraction paddy DNA |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109439657A (en) * | 2018-12-26 | 2019-03-08 | 青岛袁策集团有限公司 | A kind of rice leaf DNA rapid extracting method |
| CN109897886A (en) * | 2019-04-16 | 2019-06-18 | 安徽省农业科学院水稻研究所 | A kind of rice plumule Miho Dockyard NA template preparation method and PCR method for PCR amplification |
| CN110106066A (en) * | 2019-06-14 | 2019-08-09 | 华南农业大学 | A kind of extracting method of the paddy DNA sample suitable for high-throughput Genotyping |
-
2017
- 2017-12-30 CN CN201711498735.1A patent/CN108531546A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109439657A (en) * | 2018-12-26 | 2019-03-08 | 青岛袁策集团有限公司 | A kind of rice leaf DNA rapid extracting method |
| CN109897886A (en) * | 2019-04-16 | 2019-06-18 | 安徽省农业科学院水稻研究所 | A kind of rice plumule Miho Dockyard NA template preparation method and PCR method for PCR amplification |
| CN110106066A (en) * | 2019-06-14 | 2019-08-09 | 华南农业大学 | A kind of extracting method of the paddy DNA sample suitable for high-throughput Genotyping |
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Application publication date: 20180914 |
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