CN106047658A - Production method and application of glass microscale aspirator - Google Patents
Production method and application of glass microscale aspirator Download PDFInfo
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- CN106047658A CN106047658A CN201610409420.4A CN201610409420A CN106047658A CN 106047658 A CN106047658 A CN 106047658A CN 201610409420 A CN201610409420 A CN 201610409420A CN 106047658 A CN106047658 A CN 106047658A
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- 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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Abstract
The invention discloses a production method and application of a glass microscale aspirator. The production method includes: heating a 1mm glass capillary to soften the same, pulling open two ends, grinding the pointed end formed by fracture into an opening with the diameter being 25-30 micrometers, connecting the non-pointed end with a soft rubber tube, and sealing the tail end of the soft rubber tube to obtain the glass microscale aspirator. The glass microscale aspirator can separate microscale liquid tissue in a microscopy manner. The aspirator is used to separate high-purity cabbage type rape liquid endosperm and a liquid endosperm genome DNA extraction method are disclosed at the same time, and accordingly high-purity materials and DNA can be provided for the researches of the development of triploid endosperm and the interaction mechanism between endosperm embryo.
Description
Technical field
The invention belongs to micrurgy and technical field of molecular biology.It is more particularly to a kind of glass micro suction dispenser
Preparation method and application.
Background technology
Brassica campestris L is the big oil crop in third place in the world, the fifth-largest agriculture after Oryza sativa L., Semen Tritici aestivi, Semen Maydis and Semen sojae atricolor of the Ye Shi China
Crop.In addition to as the primary raw material of edible oil, Brassica campestris L is also that the important raw material of industry and Major Members state of European Union can be again
The primary biological resource of the raw energy.The cultivated area of China's Brassica campestris L and about the 30% of the total output Jun Zhan world, its not only relation
To the increasing income of adjustment, optimization and nearly hundred million peasants of China's crop mix, be also related to meet living standards of the people improve, meals
National food security needs is improved and ensured to structure, therefore, strengthens Rape-seed production significant.
Brassica campestris L belongs to Angiospermae (Angiospermae) Brassica genus (Brassicaceae) plant.Cabbage type rape
(Brassica napus L.) is allodiploid (AACC) crop.Its formation is before about 7500, two ancestors
Kind of Chinese cabbage (AA) and Caulis et Folium Brassicae capitatae (CC) are by hybridizing then formation after polyploidization.Therefore cabbage type rape is also research genome
The ideal material of Polyploid evolution.
The growth of angiosperm seed is by double fertilization: one of them spermatid and ovum merge generation and close
Son, develops into embryo afterwards；Another spermatid and diploid central cell merge and form triploid fertilization polar core, finally grow
Become endosperm.Endosperm is wrapped in embryo, but is coated by integument.There is the phase of endosperm development and fetal development in the growth course of seed
Synergism mutually.The exception of endosperm development often leads to the stagnation of fetal development, implies its important in Seed development
Property.There are some researches show, the oil content of model plant arabidopsis and cabbage type rape seed is affected by maternal effect.And parent
Effect can be divided into three kinds of different types: cytoplasmic inheritance, endosperm nucleus and maternal phenotype.The endosperm of brassica plant is recognized
For take part in nutrient substance by vascular tissue transport from maternal plant to embryo.Experimentation again shows that endosperm can also
Control seed size.Additionally, some are the most active with the expression of lipid and carbohydrate metabolism related gene in endosperm.Endosperm exists
The transport protein of different metabolic product take part in oil and fat accumulation process in embryo development procedure.Similarly, the generation of lipid is stored
Thank and decompose also to be regulated and controled by the part of endosperm.Except providing nutrition to embryo, maternal tissue's endosperm is also considered as apparent tune
The place of control.In endosperm, some genes (including overwhelming majority storage protein and starch synthase gene) of predominant expression are in Oryza sativa L.
Endosperm often occurs demethylation.Therefore, the mechanism of research endosperm development and the interaction of endosperm and embryo are in oil content etc.
Effect in Main Agronomic Characters is particularly important.But the liquid endosperm not a duck soup of separating high-purity, because endosperm quilt
The least and by circle integument is wrapped up, and the most adjacent with developmental embryo.
There is presently no suitable method to ensure separating high-purity liquid endosperm and to carry out high concentration, high quality DNA
Extracting, the application is by setting up the technology of separating high-purity liquid endosperm quickly and efficiently and the liquid endosperm genome of improvement
DNA extraction technique, the research for the interaction Regulation Mechanism between the growth of follow-up endosperm and endosperm and embryo is laid a good foundation.
Summary of the invention
The present invention seeks to there are provided the preparation method of a kind of glass micro suction dispenser, material is easy to get, and method is simple,
The glass micro suction dispenser of preparation has relatively broad application in micro-separation trace liquid texture.
Further object is that the application providing a kind of glass micro suction dispenser, this aspirator can be used for showing
Differential is from trace liquid texture.
The present invention seeks to there are provided a kind of efficient high-purity Brassica campestris L liquid endosperm separation method, after the method is
The continuous research carried out in terms of endosperm provides the foundation.
A further object of the invention is to there are provided the extracting method of a kind of Brassica campestris L endosperm genes group DNA.The method
High concentration, high-quality genomic DNA can be obtained, meet the demand of follow-up molecular biology experiment.
In order to realize above-mentioned purpose, the present invention uses techniques below measure:
A kind of preparation method of glass micro suction dispenser, including:
After the heating deliquescing of 1mm capillary glass tube, being pulled open from two ends lentamente by capillary tube, capillary tube eventual failure is formed non-
The thinnest tip, forms the opening of a diameter of 25～30 μm by tip polishing, and the one end at non-tip is connected with band tubing, rubber
The end seal of flexible pipe, obtains glass micro suction dispenser.
Preferably, the end of band tubing is sealed by the glass stopper fired by capillary tube.
The application in micro-separation trace liquid texture of a kind of glass micro suction dispenser, including needing in prior art to inhale
The operation taking trace liquid texture all may utilize the glass micro suction dispenser that the present invention provides.
A kind of efficient high-purity Brassica campestris L liquid endosperm separation method, including:
Select cabbage type rape Post flowering ovule volume sizing time ovule as separate endosperm material, ovule away from
The tip position of embryo utilizes draw point to punch, and after puncturing blastular, is rapidly inserted at punching by glass micro suction dispenser, slowly draws
Endosperm, is then transferred in the EP pipe being put on ice for；The endosperm microscopy drawn observes endosperm purity.
Above-described top is the top in morphology；
The process described above, whole process is carried out on ice, and the time controls in 20s.
Described material is the cabbage type rape Post flowering ovule of the 25-30 days.
In schemes described above, during punching, it is sure not to punch, punctures blastular；Punching is sure not to beat on embryo.
A kind of extracting method of Brassica campestris L endosperm genes group DNA, including:
With CTAB-free buffer (200mmol/L Tris-HCl (pH=8.0), 50mmol/L EDTA, 250mmol/L
NaCl, 1% β-mercaptoethanol) liquid endosperm of separation is washed；Then, with CTAB extract Eddy diffusion
Albuminous cell after washing, then in EP pipe, place 3～5 5mm steel balls, rapidly EP pipe is put into liquid nitrogen freezing.Put again
Put and thaw at room temperature.Wait to thaw to the when of forming mixture of ice and water, EP pipe is placed on tissue grinder instrument concussion 25～30
Second, use traditional CTAB method to extract genomic DNA subsequently.
Compared with prior art, it is an advantage of the current invention that:
Due to cabbage type rape liquid endosperm be coated in little and also inside the integument of circle, and itself also with grow
Embryo is the most adjacent, therefore isolates high-purity liquid endosperm and has difficulties.The mode of conventionally employed extruding obtains liquid endosperm,
But the method easily causes the pollution of its hetero-organization, and this pollution is very difficult to remove.The present invention is first from cabbage type rape ZY036
Isolated high-purity liquid endosperm in 25 days ovules of Post flowering.The liquid endosperm genome of first passage of the present invention improvement
DNA extraction method obtains the genomic DNA of high-quality, high concentration.In a word, this invention is that carrying out of later stage research has been established relatively
Good basis.
Seed, as the important food source of the mankind, has very important effect.Exist between Triploid endosperm and embryo
The interaction of interwoveness.Although most of dicotyledon endosperm are finally degraded, and the cotyledon institute that its nutrition is expanded
Absorb, finally leave behind the thinnest one layer.But the defect of endosperm all can cause fetal development to be stagnated.Furthermore, monocotyledon
Endosperm account for mature seed the overwhelming majority.As can be seen here, the research of endosperm is extremely necessary.But due to early stage liquid embryo
Within breast is coated in little integument, and against developmental embryo, manufacture difficulty for separating high-purity endosperm.In the present invention
The research in plant, particularly important crops of the high-purity liquid endosperm isolation technics will play a significant role.
At biological research fields, some material is little, few or buried at other organization internals.Accordingly, it would be desirable to such as glass
The utensil of micro suction dispenser operates, thus obtains highly purified enough experiment materials, establishes for carrying out of subsequent experimental
Fixed basis.The present invention provides glass micro suction dispenser to prepare by hand, and method is easy, feasible.And operate this glass micropipette
Device collection material is simple, convenient.Provide convenience for carrying out the collection of similar minor material.
Accompanying drawing explanation
The preparation process schematic diagram of Fig. 1 glass micro suction dispenser.
The form of Fig. 2 different development stage angle fruit and the form schematic diagram of different development stage ovule.
Wherein in Fig. 2, A is the form of different development stage angle fruit；In Fig. 2, B is the form of different development stage ovule.
Fig. 3 is the Post flowering ovule schematic diagram of the 25th day；
Wherein in Fig. 3, A is the form of 25 days embryos of Post flowering；In Fig. 3, B is that 25 days embryos of Post flowering are at 25 days embryos of Post flowering
Relative position in pearl and shared space size, the part on black dotted lines is safety hit bore region；；In Fig. 3, C is Post flowering
25 days ovule longitudinal cutting structure schematic diagrams.
Fig. 4 separation liquid endosperm and the flow process of DNA extracting thereof.
The form of the endosperm that Fig. 5 separates and purity detecting schematic diagram.
The Brassica campestris L liquid endosperm genomic DNA schematic diagram of Fig. 6 electrophoresis detection distinct methods extracting.
Wherein in Fig. 6, A is the Brassica campestris L liquid endosperm genomic DNA utilizing commercial kit to extract
The Brassica campestris L liquid endosperm genomic DNA of the method extracting that B provides for the present invention in Fig. 6.
Detailed description of the invention
Technical scheme of the present invention, if not otherwise specified, is the ordinary skill in the art.Described reagent or material,
If not otherwise specified, commercial channel is derived from.The embodiment of the present invention, as a example by cabbage type rape ZY036, illustrates.
Embodiment 1.
A kind of preparation method of glass micro suction dispenser, including:
1mm capillary glass tube heats on alcohol burner flame, treats capillary tube heat affected zone deliquescing, slowly from capillary tube two ends
Pulling open, capillary tube eventual failure forms the thinnest tip, and the most advanced and sophisticated polishing formed is formed the opening of a diameter of 25～30 μm,
Being connected with band tubing the one end at non-tip, the end glass stopper of band tubing seals, and obtains glass micro suction dispenser
(Fig. 1).
Described glass stopper is that capillary tube is fired.
Embodiment 2:
Determine the developmental stage of ovule for separating liquid endosperm
The flower using fine rule to align the cabbage type rape ZY036 bloomed in the present embodiment is marked.Subsequently, take away respectively
The angle fruit spending latter 8 days, 16 days, 22 days, 24 days, 25 days, 26 days and 28 days is observed, and finds that Pod length is about at Post flowering
Sizing (A in Fig. 2) in 25 days.Additionally, separate the ovule of different time sections under anatomical lens, then observe under Stereo microscope,
Finding that ovule volume is the fastest in Post flowering change in 8 days to 22 days, when the 25th day, ovule volume finalized the design almost (B in Fig. 2).Cause
This, the final selection Post flowering ovule of 25 days is as the material of separation endosperm, for following example.
Embodiment 3:
Select the punch position that cabbage type rape ZY036 ovule surface is optimal
Utilize stripping pin to separate the embryo in ZY036 25DAF ovule under inverted microscope (OLYMPUS IX71), and see
Examining its form, the embryo specifying 25DAF ZY036 is torpedo embryonic stage (torpedo stage) (A in Fig. 3)；According to
ZY036 25DAF ovule and the size of embryo and Morphologic Characteristics, utilize Photoshop simulation 25DAF embryo at whole embryo
Position in pearl and taken up space (C in B and Fig. 3 in Fig. 3), clearly on ovule surface away from embryo position be punching optimal
Rational position (more than the black dotted lines shown in B for punching safety zone in Fig. 3).
Embodiment 4:
A kind of efficient high-purity Brassica campestris L liquid endosperm separation method, including:
The surface punching utilizing the draw point top away from embryo on cabbage type rape ovule surface under anatomical lens (is punched
Time, it is sure not to punch, punctures blastular), after puncturing blastular, (hole beaten guarantees in the safety zone shown in Fig. 2 B, is sure not to beat
On embryo, otherwise can cause the pollution of its hetero-organization), glass micro suction dispenser embodiment 1 prepared is rapidly inserted at punching
(extruding band tubing, the gas inside release before inserting), slowly draws endosperm, is then transferred to the EP pipe being put on ice for
In.
The most whole process is carried out on ice, and the time controls in 20s, and the endosperm microscopy of absorption is observed without other cells dirty
Dye (Fig. 5).
Fig. 4 is asked for an interview in whole operating process.
Embodiment 5:
A kind of extracting method of Brassica campestris L endosperm genes group DNA, including:
With 1.5ml CTAB-free buffer (200mmol/L Tris-HCl (pH=8.0), 50mmol/L EDTA,
250mmol/L NaCl, 1% β-mercaptoethanol) liquid endosperm that embodiment 4 separates is washed: i.e. utilize 1ml
(blue rifle head carries out enlarging with shears to eppendorf pipettor in advance, prevents sample from blocking during suction is beaten or staving endosperm
Cell) inhale gently and play mixing, then inside the refrigerated centrifuger of 4 DEG C of pre-coolings in advance, it is centrifuged 4min with 1100rpm, abandons supernatant；
Then, the albuminous cell after washing with 750 μ l CTAB extract Eddy diffusions, then in EP pipe, place 5 5mm steel balls, rapidly
EP pipe is put into liquid nitrogen freezing.Place it in again in room temperature and thaw.Wait to thaw to the when of forming mixture of ice and water, EP is managed
Shake 30 seconds in being positioned over SK450 type vibration-type mixer (Shanghai Su Kai Chemical Co., Ltd.), use traditional CTAB subsequently
Method extracts genomic DNA, and final acquisition 200 μ l, at concentrations up to 2.83 μ g/ μ l, the preferable genomic DNA of its integrity
(Fig. 6 B).
With commercial kit (AxyPrepTMMultisource Genomic DNA Miniprep Kit) to embodiment
The liquid endosperm of 4 preparations extracts DNA.The DNA integrity degree obtained is preferable, but concentration low (≤30ng/ μ l) (Fig. 6 A).
Claims (4)
1. a preparation method for glass micro suction dispenser, including:
After the heating deliquescing of 1mm capillary glass tube, being pulled open from two ends lentamente by capillary tube, capillary tube eventual failure is formed very
Thin tip, forms the opening of a diameter of 25 ~ 30 μm by tip polishing, and the one end at non-tip is connected with band tubing, and rubber is soft
The end seal of pipe, obtains glass micro suction dispenser.
2. the glass micro suction dispenser that the method described in claim 1 prepares application in micro-separation trace liquid texture.
Application the most according to claim 2, it is characterised in that:
Ovule when selecting cabbage type rape Post flowering ovule volume to shape is as the material of separation endosperm, at ovule away from embryo
Tip position utilize draw point to punch, after puncturing blastular, glass micro suction dispenser is rapidly inserted at punching, slowly draws embryo
Breast, is then transferred in the EP pipe being put on ice for；The endosperm microscopy observation of liquid state endosperm purity drawn；
Described method, whole process is carried out on ice, and the time controls in 20s；During punching, it is sure not to punch, punctures blastular i.e.
Can；Punching is sure not to beat on embryo.
Application the most according to claim 3, it is characterised in that:
With CTAB-free buffer (200 mmol/L Tris-HCl (pH=8.0), 50 mmol/L EDTA, 250 mmol/
L NaCl, 1% β-mercaptoethanol) liquid endosperm of separation is washed；Then, with CTAB extract again
Suspend the albuminous cell after washing, then places 3 ~ 55 mm steel balls in EP pipe, and EP pipe is put into rapidly liquid nitrogen freezing, then will
Its placement is thawed at room temperature；Wait thaw to formed mixture of ice and water when, EP pipe is placed on tissue grinder instrument concussion 25 ~
30 seconds, use traditional CTAB method to extract genomic DNA subsequently.
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| CN201610409420.4A CN106047658A (en) | 2016-06-13 | 2016-06-13 | Production method and application of glass microscale aspirator |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109355178A (en) * | 2018-10-24 | 2019-02-19 | 四川大学华西医院 | Continuous volume gradient capillary digital PCR device and using method thereof |
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| CN2798089Y (en) * | 2005-04-14 | 2006-07-19 | 李国华 | Apparatus for diaphragm forceps intracellular dialysis |
| CN101167657A (en) * | 2007-12-10 | 2008-04-30 | 北京师范大学 | Mouse cerebrospinal fluid microscale sampler and its special-purpose manufacturing equipment and using method |
| CN201186217Y (en) * | 2008-03-12 | 2009-01-28 | 徐州医学院 | General accurate microburet for acid and alkali |
| CN102345994A (en) * | 2011-08-29 | 2012-02-08 | 华南理工大学 | Composite liquid absorption core of heat dissipation heat pipe and manufacture method thereof |
| CN106000496A (en) * | 2016-06-07 | 2016-10-12 | 中国农业科学院油料作物研究所 | Preparation method and application of glass micro-liquid sucking device |
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2016
- 2016-06-13 CN CN201610409420.4A patent/CN106047658A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2798089Y (en) * | 2005-04-14 | 2006-07-19 | 李国华 | Apparatus for diaphragm forceps intracellular dialysis |
| CN101167657A (en) * | 2007-12-10 | 2008-04-30 | 北京师范大学 | Mouse cerebrospinal fluid microscale sampler and its special-purpose manufacturing equipment and using method |
| CN201186217Y (en) * | 2008-03-12 | 2009-01-28 | 徐州医学院 | General accurate microburet for acid and alkali |
| CN102345994A (en) * | 2011-08-29 | 2012-02-08 | 华南理工大学 | Composite liquid absorption core of heat dissipation heat pipe and manufacture method thereof |
| CN106000496A (en) * | 2016-06-07 | 2016-10-12 | 中国农业科学院油料作物研究所 | Preparation method and application of glass micro-liquid sucking device |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109355178A (en) * | 2018-10-24 | 2019-02-19 | 四川大学华西医院 | Continuous volume gradient capillary digital PCR device and using method thereof |
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Application publication date: 20161026 |