CN109883797A - A kind of method of easy long-tail anchovy chromosome preparation - Google Patents
A kind of method of easy long-tail anchovy chromosome preparation Download PDFInfo
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Abstract
The present invention relates to a kind of methods of easy long-tail anchovy chromosome preparation, belong to cytogenetics field, which comprises 1) long-tail anchovy sampling device and operation；2) sample is hypotonic, fixes and dissociates；3) sheet devices and operation are dripped；4) it dyes；The method that the present invention prepares long-tail anchovy division phases chromosome is simple and fast, and whole flow process only needs 2~3 hours, and device can flexible assembling, and it is easy to operate, it can carry out whenever and wherever possible, be especially suitable for the occasion that farm, seashore base etc. do not have laboratory condition, the chromosome sectioning of preparation is completely apparent.
Description
Technical field
The invention belongs to cytogenetics field, it is specifically related to a kind of easy long-tail anchovy chromosome preparation facilities and operation
Method.
Background technique
Chromosome is the carrier of inhereditary material, studies species chromosome, not only can inquire into its classification position and system is drilled
Change, it helps the identification and colony assay, development and utilization and genetic breeding to resource of nearly edge species have important meaning.
Chromosome analysis or an important means of various animal diagnosis of genetic disorders and character variation research, even more genome are grasped
Make a special kind of skill that breeding must be grasped.Culture fishery also usually need to be grasped the genomes of cultivated species at and structure
Etc. information.Especially in recent years, the phenomenon that marine animal asyngamy, has attracted the concern of a large amount of experts, the spy of Sex Determination Mechanism
Begging for also is always one of most attractive and most popular field in life science, if there are sex chromosome is to study
The problem of personnel most want to know about.20th century first half leaf, it is cytogenetic studies have shown that sex chromosome is between animal species,
Even all there is variations for very close inter-species, it means that evolution of sex chromosomes is very fast.Genetics research later also found,
Even if in some chromosomes obvious identical inter-species, Sex Determination Mechanism is also dramatically different, or even also studies have found that gender is determined
Determine mechanism all to have differences in kind.To carry out accurate CYTOGENETIC ANALYSIS OF ONE, chromosome specimen preparation is most important
One link, key are piece of drawing materials and drip, and excellent chromosome sectioning is the basic of cytogenetical study.
1966, (Ojima Y S, Hitotsumachi S, the Makino.Cytogentic studies in such as Ojima
Lower vertebrates.Proc Jap Acad, 1966,42 (1): 62-66) for the first time using airing film-making obtain at
Function, all in this way, but unmodified this preparation method is not again because fill specifically for current chromosome sectioning
It sets, and flaking step is cumbersome, is difficult that clear and well dispersed piece of chromosome morphology is made, is not able to satisfy chromosome correlation point
The requirement of analysis.
Long-tail anchovy (Coilia mystus) belongs to river mouth migration fishes, is the main of the rivers mouth such as the Changjiang river, the Zhujiang River, Min River
Economic fish, fishing season season yield is very high, and entrance of Changjiang River annual output is once up to 5000t or more.Just the prelarva of hatching soon is just in river
It is fattened at the deep water in river mouth, returns later marine, next year reaches sexal maturity, and general female fish length is slightly larger than milter.During fattening
The speed of growth is most fast, and Cell division in vivo Reproductive activity is strong, is suitble to prepare chromosome, but long-tail anchovy has a feature --- and property is strong (to answer
Sharp reaction is very strong), dead quickly once being caught and pulling the water surface out, histiocytic division and proliferation ability also disappears soon
It loses, even if fishing is transferred in water body on the bank, the time-to-live is also very short, is usually no more than half an hour, it is non-that chromosome prepares difficulty
Chang great, this is also so far, there is not yet the reason of long-tail anchovy chromosome is reported.Related specy, such as knife long-tailed anchovy (Coilia
Nasus) (Xu Shijie, Li Yuanyuan, Fu Guanbao, Wu Haowei, Wang Qian, Liu Qigen, Qu Xiancheng.Knife long-tailed anchovy chromosome karyotype analysis, Guangdong
Agricultural sciences, the 7th phase of volume 41 in 2014,155-157 pages) and brachygnathia long-tailed anchovy (Coilia brachygnathus) (Hong Hanyun, week
Newly-risen sun.The caryogram and its ZZ-ZO sex chromosome of brachygnathia long-tailed anchovy, heredity, the 4th phase of volume 6 in 1984,12-14 pages) have chromosome report
Road, and all think that male and female chromosome has differences, sex chromosome is reported as ZZ-ZO type, i.e. male chromosome number is 2n
=47, x chromosome number is 2n=48 item.Both fish chromosomes are prepared again all using nephridial tissue as material, wherein knife long-tailed anchovy
Prepare that (actual result is only limitted to nephrocyte, and does not refer to the gill using the gill-nephrocyte phytohemagglutinin-colchicine method
Cell situation), brachygnathia long-tailed anchovy then according to nephridial tissue cell culture, be air-dried or the preparation of flame seasoning, the two has nephridial tissue
Cell culture link, required time is long and complex steps, final result also need further to be verified.
Therefore, the preparation of long-tail anchovy chromosome need to fully consider the biological characteristics of itself, and materials and pre-processing can only be just
Approximately principle still keeps certain vigor intensity to ensure long-tail anchovy histocyte in colchicine treatment process, can prepare
Chromosome.However, study condition is mostly simple and crude on nearest seashore or fishing fishing boat, do not have expensive and advanced analyzer
Device, there is an urgent need to a kind of simple easily chromosome preparation facilities and the simple methods of operating procedure.
Summary of the invention
The technical problem to be solved in the present invention is that a kind of method of easy long-tail anchovy chromosome preparation is provided, it is described to pass through
Improvement to existing marine animal method of chromosome preparation designs an easy long-tail anchovy chromosome preparation facilities, with most economical
But most convenient and fast method prepares a large amount of well dispersed division phases chromosome.It can quickly be made using the method for the present invention
Standby long-tail anchovy adult fish division phases chromosome a large amount of out is to meet species Germplasm Identification, ploidy analysis, the situ Analysis
Etc. the demand in relation to chromosome research.The limitation that materials site condition and species biological nature can be overcome, since obtaining tissue
To observation result, it is only necessary to which 2~3 hours, device was easily obtained and assembled, and operating procedure is simple, and it is strong that chromosome preparation can be adjusted flexibly
Degree, can by the sea, river mouth nearby even carries out on fishing boat at any time.
The present invention specifically includes following four steps and operations method:
A kind of method of easy long-tail anchovy chromosome preparation, which comprises 1) long-tail anchovy sampling device and operation；2) sample
Product are hypotonic, fix and dissociate；3) sheet devices and operation are dripped；4) it dyes；
The long-tail anchovy sampling device: the long-tail anchovy sampling device includes a bubble chamber with cover, and bubble chamber internal volume is extremely
Few 5L, bubble chamber wall thickness 2cm~8cm have preferable compression strength, and foam case lid is smooth, rectangular to be preferred, and space is steeped in case
Foam plate is separated into 2 parts, and a part is storing part, places materials utensil, and another part is freeze space, places ice bag or ice chest,
Place one bottle of 20~100ml colchicine mother liquor, empty 100~250ml fixer bottle, 10~50ml dissociation in the area
Liquid bottle, 1 10ml centrifuge tube shelf；All reagent bottles are all sat into the sample sack of close size in case, and 1~2 is placed in sample sack
On the one hand disposable dropper a 2ml or 3ml with a scale can prevent the pollution between reagent for drawing related solution；It is another
Aspect can prevent impact failure between glass reagent bottle, and key also facilitates reagent to take；
Further, the materials utensil is scissors, tweezers, scalpel, 10ml centrifuge tube, 1.5ml centrifuge tube, 1
1.5ml centrifuge tube shelf, 1 10ml centrifuge tube shelf, 500ml analyze pure 1 bottle of glacial acetic acid, 500ml analyzes pure dehydrated alcohol or methanol 1
Bottle, a distilled water or ultrapure water wash bottle, 1 bottle of 250-500ml0.075MKCl solution, a dyeing liquid kit, the dyeing
Liquid kit includes 10-50ml dyeing liquor mother liquor, 100-500ml PBS phosphate buffer, 1 50ml with range scale flat
Centrifuge tube and 2ml or 3ml Dispette with a scale further include 50ml flat centrifuge tube with a scale in the materials utensil
3, the clean chromosome of a box prepare dedicated glass slide, a multiple tracks timer, 1 packet rubber band and other small articles；
The operation of long-tail anchovy materials is fishing long-tail anchovy, and long-tail anchovy one goes out after the water surface, and it is full to select sexual gland rapidly, still vibrant, body
The adult fish of long 12~16cm opens the gill cover with tweezers, and the clip part gill filament, investment shifts to an earlier date prepared 0.04% colchicum at once
In plain solution, 0.04% colchicine solution is placed on 10ml containing 50% local filtering sea, the colchicine solution
In centrifuge tube, the 10ml centrifuge tube is placed on 10ml rack for test tube, and is placed on bubble chamber and is covered 30min；At this point, by storing part
Tool taken out from bubble chamber, be placed on bubble chamber and cover, foam case lid covers on bubble chamber, keep freezing low temp area shape
State；During colchicine processing, it is long to measure and record the long-tail anchovy body being sampled, and dissects sexual gland or squeezes abdomen, observes reproduction
Liquid status is to identify long-tail anchovy gender；
Further, it is the cleaning for keeping foam case lid, covers one layer of freshness protection package before each use on bubble chamber case lid.
The sample is hypotonic, fixes and dissociates: fixed and dissociation operation is carried out in the freeze space of long-tail anchovy sampling device；
After colchicine handles tissue sample, sample is placed in hypotonic solution, 45~60min of Hypotonic treatment；Period
Centrifuge tube turn upside down for several times, it is ensured that hypotonic abundant；Hypotonic period configuration fixer and dissociation solution are put into the freeze space of bubble chamber
Refrigeration；The fixer is that the volume ratio of dehydrated alcohol or methanol and glacial acetic acid is 3:1；The dissociation solution is glacial acetic acid and distillation
Water volume ratio 1:1；
At this moment 10ml centrifuge tube shelf and 1.5ml centrifuge tube shelf are all put into freeze space pre-cooling；After tissue sample is hypotonic,
Hypotonic solution is discarded, fixed solution, fixed 15min is added, fixed sample cell is inserted on the rack for test tube of freeze space, covers bubble chamber
Lid, it is ensured that entire fixation procedure is all carried out in freeze space；Fixed liquid measure need to be totally submerged tissue.After 15min, fixation is discarded
Liquid, the fixed solution of the pre-cooling then more renewed fix 15min again, and fixed sample cell is still inserted into freeze space rack for test tube
On, foam case lid is covered, is repeated this fixing step 3~4 times, sample is fixed abundant；By tissue sample be put into 1.5ml pre-cooling from
In heart pipe, added with the dissociation solution of 0.5~1.5ml pre-cooling in centrifuge tube, it is then placed in freeze space or 4 DEG C of refrigerators and dissociates；
Further, the hypotonic solution is 0.05mol/L KCl.
Further, it in dissociation process, rocks or shakes dissociation tube wall or disposable dropper piping and druming dissociation solution can be effective
Accelerate the dissociation of sample tissue cell.
The drop sheet devices and operation, drop sheet devices include 25~40cm ruler, 1 handle, the disposable dropper of 1 parcel, electric furnace or
Electromagnetic oven 1, one piece of 15~25cm × 30~50cm size heated sheet, straightening ruler and set up heated sheet include brick or stone
Weight several piece including block can accommodate iron frame 1 of electric furnace or electromagnetic oven；
Operation: looking for the table top of a liftoff 1~1.5m high first, by weight pressure of the one end of ruler including brick
It firmly or fixes, the other end vacantly spreads from the table, and the underface of ruler extension end is the center of electric furnace or electromagnetic oven, in electromagnetic oven
Or hot plate of feeling better is mounted right above electric furnace, heated sheet is heated evenly heated sheet and unlikely preferably above the stove at 2~10cm
In fragmentation, open heating device it is suitable to heated plate temperature when can carry out heat drop piece, when non-transformer, carries out cold drop piece；
The dyeing of the chromosome sectioning: being configured to 10% Giemsa stain, and the drop piece dried is laid flat with disposable
Dropper is drawn 1~1.5ml dye liquor and is gently dripped on the glass slide that drop has sample, and pulls liquid by surface tension of liquid with suction pipe
Drop top makes it be paved with glass slide, guarantees that sample is covered by dye liquor entirely, after dyeing 30min, discards dye liquor, thread cleaning carries glass
On piece remaining 5~10s of dye liquor is placed on 45~85 ° of angle specimen face sides of glass slide, and upper toilet paper water suction is padded in bottom, and film-making is dried in the air
Microscopy is observed for microscope after dry and is taken pictures.The chromosome sectioning of no dyeing can be not only used for chromosome band type analysis, and can
To be used for FISH analysis.
The present invention compared with the prior art the utility model has the advantages that
1. the present invention preparation long-tail anchovy division phases chromosome method it is simple and fast, device can flexible assembling, and easily behaviour
Make, the chromosome sectioning of preparation is completely apparent, convenient for observation and counts, circling around 3 drop cell circles can be completed one
The observation of chromosome sectioning will not miss scattered in the sample and good division phases chromosome of form.The chromosome of preparation
Film-making is placed on can long-term preservation (several months to several years) in slice box.
2. can be repeated several times preparation, whole flow process is only needed 2~3 hours.It can carry out whenever and wherever possible, especially suitable farm,
Seashore base etc. does not have the occasion of laboratory condition.This method cannot be only used for the preparation of long-tail anchovy chromosome, apply also for other
Various marine animal chromosome preparations, biopsy sample, which is gathered materials on the spot, directly carries out chromosome preparation, increases split coil method without early period
Step chooses organism and divides vigorous tissue.
3. few using sample size needed for present apparatus film-making, 3 drop of Dispette drop can utmostly guarantee to be drawn materials
The histiocytic activity of biological object.Preparation of reagents is reduced to centrifuge tube and suction pipe with a scale and measures, centrifuge tube and suction pipe
It is plastic products, it is non-breakable, easy to operate and low in cost.
4. distinguishing male and female according to the state of long-tail anchovy adult fish body size and gonad trickle, easy to operate, judgement
Accurately, there is practicability.Using dotted male Y chromosome can be obviously observed in milter chromosome prepared by the present invention, fill
Dividing proves that long-tail anchovy Sex Determination mode is XX/XY type.
Detailed description of the invention
Fig. 1 heat drips piece diagram；
Fig. 2 hero long-tail anchovy division phases chromosome (arrow instruction sex chromosome).
Specific embodiment
Below by specific embodiment combination attached drawing, the present invention is described in detail, is that most preferred embodiment is specifically grasped below
Make technical process, protection scope of the present invention is not limited in any form by embodiment.
Embodiment:
In August, 2018, Shanghai Chongming island, fishing boat sea fish long-tail anchovy.Death is easy for since long-tail anchovy fish one goes out the water surface
Characteristic, the chromosome that prepare long-tail anchovy division phases must be rapid, we just will dye system before fishing long-tail anchovy
Standby device and various reagents consumptive material are ready.Select 16cm body raun 1 long according to size, the long milter of 13cm body one, two
Tail fish belly portion is full, and has reproduction liquid to flow out from cloacal aperture, and emulsion form is milter, and particle is significantly raun.Then it selects most
The long-tail anchovy gill tissue easily obtained and isozyme carry out that colchicine effect, different gradient KCl solution is hypotonic, 3-4 Carnoy
Family name's fixer is fixed, heat drop piece method drips piece and dyeing, is successfully prepared the division phases chromosome of long-tail anchovy, and have found milter
Special-shaped sex chromosome.
It is as follows to complete the concrete operations of aforesaid operations process:
Easy long-tail anchovy chromosome sectioning device and operating method, step include: 1) long-tail anchovy sampling device and operation；2)
Sample is hypotonic, fixes and dissociates；3) sheet devices and operation are dripped；4) it dyes；
Be ready in advance it is clean, without greasy dirt, do not hang the chromosome sectioning glass slide of water, mounted box is spare, avoids pollution.In advance
It is put with the clean mineral water bottle of 500~600ml and freezes ice making 2, bottle in -80 DEG C of refrigerators.Prepare the bubble chamber of 10L or so, it will
2 ice chests are put into case, form freezing region.By the colchicine mother liquor of preparation, the fixer reagent bottle of KCl hypotonic medium and sky
And dehydrated alcohol is put into the pre-cooling of freezing region.Reagent bottle is packed into the sealed bag of suitable size, prevents from colliding between reagent bottle
The pollution of damaged and solution.It is also placed in the sealed bag of corresponding reagent after taking the Mark labels of disposable dropper of reagent.Examination
It is separated between agent bottle with 10ml centrifuge tube shelf and 1.5ml centrifugation tube sheet.Before catching long-tail anchovy, 0.04% colchicine is first prepared
Working solution (50% seawater containing 0.04% colchicine) is subject in 10ml centrifuge tube and is centrifuged tube wall high scale, tool
Gymnastics is made: instilling 0.4% colchicine mother liquor of 1ml, adds 4.5ml ultrapure water and the corresponding sea area filtering sea of 5ml to always
Volume 10ml.Rapid clip long-tail anchovy gill tissue and isozyme are put into 0.04% colchicine working solution and handle 40-45 minutes.
It is 0.05mol/ that the KCl hypotonic solution of the 0.075mol/L prepared is diluted to concentration with ultrapure water during acting on by colchicine
L and 0.0375mol/L forms 3 gradients, and 10ml of being still subject to is centrifuged tube wall scale, is packed into 5-10ml in 1 pipe
0.075mol/L KCl is as high concentration hypotonic medium；It is respectively charged into 2 parts of 0.075mol/L KCl in 1 pipe and 1 part of pure water is prepared
At 0.05mol/L KCl solution, as middle concentration hypotonic medium；One pipe is respectively charged into 0.075mol/L KCl and each 1 part of pure water
It is configured to 0.0375mol/L KCl solution, as low concentration hypotonic medium.It will be handled through colchicine with tweezers or disposable dropper
Tissue choose, tissue is divided into three equal parts with scissors or scalpel, is respectively put into three pipe hypotonic mediums and impregnates 50 minutes, the step
It is rapid to be used to determine best hypotonic concentration, centrifuge tube can be gently turned upside down for several times during hypotonic, make histocyte is hypotonic to fill
Point.After hypotonic beginning, Ka Nuoshi (Carnoy) fixer (methanol: glacial acetic acid=3:1) is prepared with 50ml centrifuge tube with a scale
100ml is packed into the fixer reagent bottle of pre-cooling (100ml), is placed on freeze space and continues to be pre-chilled.Then according at fish length and life
Grow the gender that liquid status judges and records fish.To hypotonic medium is sucked out into waste liquid bottle with Dispette after hypotonic.Again
Fixer with the pre-cooling of Fresh is sufficiently fixed, and fixed liquid measure 2-5ml, tissue sample is completely immersed in fixer, every fixation
Replacement fixer is primary after 15min, and discarded fixer pours into waste liquid bottle, and fixation procedure is all carried out in freeze space, repeats
It is at least 3 times fixed.Sample was fixed after 1 hour, in glacial acetic acid: ultrapure water=1:1 ratio prepares dissociation solution 3ml, and 12 parts solid
1~8mm is taken in random sample product3Size tissue enters in 1.5ml centrifuge tube, sample is dissociated with the dissociation solution of 0.5ml or so, to accelerate
Sample dissociation, flicks tube wall with finger or centrifuge tube turns upside down for several times, then the sample of dissociation is placed on freeze space pre-cooling.For
It is easy to operate, when colchicine effect and Hypotonic treatment sample, sample is transferred on the bank from ship.Laboratory is not reached
When, fixed sample is stored in freezing region and is transferred in 4 DEG C of refrigerators at once after reaching laboratory.
The drop sheet devices and operation, drop sheet devices include 25~40cm ruler, 1 handle, the disposable dropper of 1 parcel, electric furnace or
Electromagnetic oven 1, one piece of 15~25cm × 30~50cm size heated sheet, straightening ruler and set up heated sheet include brick or stone
Weight several piece including block can accommodate iron frame 1 of electric furnace or electromagnetic oven.
Before dripping piece, drop sheet devices are installed by Fig. 1 heat drop piece diagram, the experiment adjustable electric furnace of indoor power is heat source, purchase
Serve when buying other expensive instruments one piece of the iron plate (area 15cm × 40cm) of packaging support with brick mount electric furnace just on
Fang Dangzuo heated sheet, from electric furnace about 8cm.The ruler that one end is vacantly laid flat is pushed down on experimental bench, ruler one end is stretched out outside experimental bench,
The distance between ruler extension end and vertical lower heated sheet are about 1.2m, first draw pure water in heated sheet with Dispette
Vertically examination drop 3 is dripped down for three positions of adjacent 2cm on central area just above ruler, according to the position of 3 water droplets on heated sheet
Determining glass slide placement position is set, and draws glass slide profile in this region in heated sheet with marking pen, wipes water droplet away with toilet paper.
When by electric furnace electrified regulation heated sheet, the filter paper of doubling is cut into angular paper by two layers of circular filter paper doubling with scissors
Piece, still have between four layers of scraps of paper part be connected to folding area be allowed to be not easy it is loose.Electric furnace heating makes iron plate surface temperature close to 60 DEG C
When, finger abdomen or the palm of the hand can park 10s or so in heated sheet center, i.e. closing power supply.Moment finger is wanted during heat drop piece
Experience heated plate temperature, be heated the too high timely closing power supply of plate temperature, and it is too low, it needs to reopen power supply heating, it is ensured that temperature is permanent
Piece is dripped again when being scheduled on 60 DEG C or so.Clean glass slide is put into the position of label, is drawn with the Dispette of pre-cooling pre-
Cold dissociation solution, rapidly vertically drip at three determining graduation positions 3 drops dissociation sample on ruler, then at once will drop
There is the glass slide of sample to be moved to the edge that iron plate is not heated, shear 4 layers of filter paper sharp corner are immersed into dropping liquid center, are borrowed
Capillary phenomenon between drainage paper layer quickly exhausts dropping liquid, and the cell in dropping liquid is pulled in drop circle, cell is made to be uniformly dispersed, together
When cell or chromosome are firmly attached on glass slide by the residual temperature on glass slide, also make in remaining a small amount of hypotonic medium
Glacial acetic acid and distilled water volatilization are clean.The label of sample group and film-making time is carried out at glass slide frosted glass with pencil.
Dyeing: it after heat drop on piece dissociation solution is thoroughly dried, moves to bubble chamber and covers and lay flat.Two tail fish of male and female totally 12 parts of samples
2 pieces are respectively dripped, altogether 24 pieces.3ml Giemsa staining liquid mother liquor is drawn with disposable dropper, until in 50ml centrifuge tube,
Using 50ml tube wall scale as reference, add at phosphate buffer to 30ml scale, is stirred evenly, be configured to the piping and druming of disposable dropper
10% Giemsa stain.1-1.5ml dye liquor is drawn with disposable dropper gently to drip on slide specimen face, and is relied on suction pipe
Surface tension of liquid, which pulls, makes it be paved with slide specimen face at the top of drop, after dyeing 30min, take glass slide to write mark with finger
At the frosted glass of label, above translation glass slide to waste liquid bottle, dye liquor is removed, and wash away remaining dye liquor 5 on glass slide with wash bottle
~10s, tiltedly according on the side of corner on 45~85 ° of angle specimen face sides of glass slide, upper toilet paper water suction is padded in bottom, it is ensured that sample surface water
Part thoroughly parches, and does not stay water stain mark.The film-making of Giemsa staining is placed on microscopically observation microscopy and is taken pictures.0.05mol/
Division phases are more in the sample of L KCl hypotonic solution processing, and chromosome morphology and dispersion degree are preferable.Fin ray dissociated cell number
It is few, be not suitable for preparation long-tail anchovy chromosome.Colchicine processing 40min or so the time is a little partially long, and chromosome condensation is serious；It is all
Experiment condition advanced optimizes, and reduces colchicine and handles the time to 30min, prepare form be more clear, elongated dyeing
Body has 1, apparent graininess sex chromosome in milter chromosome, and total chromosome number is 2n=48 item, thus proves long-tail anchovy property
Other deciding means are XX/XY type, see Fig. 2.
Claims (5)
1. a kind of method of easy long-tail anchovy chromosome preparation, it is characterised in that the described method includes: 1) long-tail anchovy sampling device and
Operation；2) sample is hypotonic, fixes and dissociates；3) sheet devices and operation are dripped；4) it dyes；
The long-tail anchovy sampling device: the long-tail anchovy sampling device include a bubble chamber with cover, bubble chamber internal volume at least 5L,
Bubble chamber wall thickness 2cm~8cm, space is separated into 2 parts in case, and a part is storing part, places materials utensil, another part
For freeze space, ice bag or ice chest are placed, which places one bottle of 20~100ml colchicine mother liquor, a 100 empty~250ml
Fixer bottle, 10~50ml dissociation solution bottle, 1 10ml centrifuge tube shelf；All reagent bottles are all sat into close size in case
1~2 2ml or 3ml disposable dropper with a scale is placed in sample sack, in sample sack；
After long-tail anchovy materials operation is catches out long-tail anchovy, full, still vibrant, the long 12~16cm of body the adult fish of sexual gland is selected rapidly,
The gill cover is opened with tweezers, the clip part gill filament, investment shifts to an earlier date in prepared 0.04% colchicine solution at once, described
0.04% colchicine solution is placed on 10ml centrifuge tube containing the local filtering sea of volume ratio 50%, the colchicine solution
In, the 10ml centrifuge tube is placed on 10ml rack for test tube, and is placed on bubble chamber and is covered 30min；At this point, by the tool of storing part
It is taken out from bubble chamber, is placed on bubble chamber and covers, foam case lid covers on bubble chamber；During colchicine processing, measurement is simultaneously
It is long to record the long-tail anchovy body being sampled, dissect sexual gland or squeezes abdomen, observes reproduction liquid status to identify long-tail anchovy gender；
The sample is hypotonic, fixes and dissociates: fixed and dissociation operation is carried out in the freeze space of long-tail anchovy sampling device；
After colchicine handles tissue sample, sample is placed in hypotonic solution, 45~60min of Hypotonic treatment；Above and below period
Reverse centrifuge tube is for several times, it is ensured that hypotonic abundant；Hypotonic period configuration fixer and dissociation solution are put into the freeze space refrigeration of bubble chamber；
The fixer is the mixed liquor that the volume ratio of dehydrated alcohol or methanol and glacial acetic acid is 3:1；The dissociation solution be glacial acetic acid with
The mixed liquor of distilled water volume ratio 1:1；
10ml centrifuge tube shelf and 1.5ml centrifuge tube shelf are put into freeze space pre-cooling；After tissue sample is hypotonic, discard hypotonic molten
Fixed solution, fixed 15min is added in liquid, and fixed sample cell is inserted on the rack for test tube of freeze space, covers foam case lid, it is ensured that whole
A fixation procedure is all carried out in freeze space；Fixed liquid measure need to be totally submerged tissue；After 15min, fixer is discarded, is then replaced
The fixer of new pre-cooling fixes 15min again, and fixed sample cell is still inserted on the rack for test tube of freeze space, covers bubble chamber
Lid repeats this fixing step 3~4 times, and sample is fixed abundant；Tissue sample is put into the centrifuge tube of 1.5ml pre-cooling, centrifuge tube
In added with 0.5~1.5ml pre-cooling dissociation solution, be then placed in freeze space or 4 DEG C of refrigerators and dissociate；
The drop sheet devices and operation: drop sheet devices include 25~40cm ruler, 1 handle, 1 parcel of disposable dropper, electric furnace or electromagnetism
One, furnace, one piece of 15~25cm × 30~50cm heated sheet, straightening ruler and set up heated sheet including brick or stone
Weight several piece or can accommodate iron frame 1 of electric furnace or electromagnetic oven；
Operation: looking for the table top of a liftoff 1~1.5m high first, one end of ruler pushed down with weight or is fixed on table top,
The other end vacantly spreads from the table, and the underface of ruler extension end is the center of electric furnace or electromagnetic oven, electromagnetic oven or electric furnace just on
Side mounts hot plate of feeling better, and heated sheet place 2~10cm above stove is heated evenly heated sheet and is unlikely to fragmentation, opens and add
Thermal can carry out heat drop piece when suitable to heated plate temperature, when non-transformer carries out cold drop piece；
The dyeing of the chromosome sectioning: being configured to 10% Giemsa stain, and the drop piece dried is laid flat with disposable dropper
It draws 1~1.5ml dye liquor gently to drip on the glass slide that drop has sample, and pulls glob top by surface tension of liquid with suction pipe
Portion makes it be paved with glass slide, guarantees that sample is covered by dye liquor entirely, after dyeing 30min, discards dye liquor, thread cleans on glass slide
Remaining 5~10s of dye liquor is placed on 45~85 ° of angle specimen face sides of glass slide, and upper toilet paper water suction is padded in bottom, after film-making is dried
Microscopy is observed for microscope and is taken pictures.
2. a kind of method of easy long-tail anchovy chromosome preparation according to claim 1, it is characterised in that the materials
Utensil is scissors, tweezers, scalpel, 10ml centrifuge tube, 1.5ml centrifuge tube, 1 1.5ml centrifuge tube shelf, 1 10ml centrifuge tube
Frame, 500ml analyze pure 1 bottle of glacial acetic acid, 500ml analyzes pure dehydrated alcohol or 1 bottle, one distilled water of methanol or ultrapure water wash bottle,
1 bottle, one dyeing liquid kit of 250-500ml 0.075M KCl solution, the dyeing liquid kit include 10-50ml dyeing
Liquid mother liquor, 100-500ml PBS phosphate buffer, the flat centrifuge tube of 1 50ml with range scale and 2ml or 3ml are with a scale
Dispette further includes the clean dyeing system of 50ml flat centrifuge tube 3 with a scale, a box in the materials utensil
Standby dedicated glass slide, a multiple tracks timer, 1 packet rubber band and other small articles.
3. a kind of method of easy long-tail anchovy chromosome preparation according to claim 1, it is characterised in that keep foam
The cleaning of case lid covers one layer of freshness protection package of covering in bubble chamber before each use.
4. a kind of method of easy long-tail anchovy chromosome preparation according to claim 1, it is characterised in that described is hypotonic
Solution is 0.05mol/L KCl, and hypotonic period turns upside down centrifuge tube for several times, it is ensured that hypotonic abundant.
5. a kind of method of easy long-tail anchovy chromosome preparation according to claim 1, it is characterised in that in dissociation process
In, dissociation tube wall or disposable dropper piping and druming dissociation solution are rocked or shaken to accelerate the dissociation of sample tissue cell.
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| CN115656169A (en) * | 2022-10-14 | 2023-01-31 | 宁波市海洋与渔业研究院 | Method for making scomberomorus niphonius chromosome karyotype diagram |
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