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1.Method of collection and treatment of animal’s sample
The data of science of heredity become increasingly important when and house in the wild animal. In this text, we have summarized some methods that can obtain sample and data simply and conveniently and effectively.
We before gathering samples, had better analyze to science of heredity that will go on that understands to some extent. However, collectors are not all researchers, when there are suitable distances with the laboratory gatheringing, the collector should often keep samples until having appropriate recipients or storing places, this requires collectors to possess certain experience.
First of all, all samples should be marked with the individual serial number, and should write that plant the names of name, gender, persons who sample and gather date, the more exhaustive the better, so that the same individual’s different type data can be quoted each other, in addition, the annotation of the geographical source is very important too, need marking out. The animal that caught as to the field, the person who has condition had better indicate place of capture and longitude and latitude in the map. Have same record and exhaustive pedigree picture to catch one kind of sources each field to housing, because the pedigree picture can be used for calculating that hand in the coefficient near.
In a word, the more detailed the background is, the better the background is, even can’t get all information listed here, also exhaustive as much as possible.
l. Measurement method of a sample
The standard that adopts in certain amounts of classification groups can be used in four following respects while measuring the method:
( 1) On taxonomy: A set of ideal measurement methods should be able to roughly rebuild shape and size of each main hardware part of organism.
( 2) Unsymmetry is analyzed: The wave of organism every part is asymmetric, perhaps indicate the evolution course is becoming unstable to a certain extent, this may be environmental pressures or near results handed in. Measuring the asymmetric opportunity body left and right sides two part should be taken into account.
( 3) Hereditary variation: At the section level, if adopt the same measurement method to the individual of the same age, probably make a variation and carry on the “ball-park” estimate to the hereditary composition in the characteristic selected. Always accomplish, so, can make geneticist to characteristic (if the intersection of reproduction and the intersection of characteristic and the intersection of and heredity any lose while being mutant) selected Monitor.
( 4) The physiology is found or recorded: Adopt the record that the standard measurement method got, perhaps will contribute to with the connected analysis of data that is got by means of others. For example, if there are records of sperm’s shape, or the infection record of the special parasite, relevance of competence that the geneticist can look for these factors in different colonies and make a variation.
1. Collection and save of 2 tissue samples
The table has listed the commonly used use in the research of science of heredity of organization by 7-1. Provide the appropriate method that is directed against different research purposes in detail as follows. Certainly, there is a disciplinary actions method to get used to respectively in each laboratory, so, this text only provides the relevant requirements kept fast, but the usability to each kind of tissue is commented to some extent, so that when the use has conflicts, can to to employ make fast judgement perhaps potential data.

Note: The figure represents the method to narrate in text in the table. A digital ” 1 ” Namely represent 1 of text. 2. The method in 1, digital ” 2 ” Namely represent 1 of text. 2. Method in 2. And so on and so forth the others.
Use tumour, hair and sperm’s sample, should pay special attention to while keeping. Use cells to train samples to reduce to the living subject or kill the reliance of the animal newly, train thing come from tumour organize cell, can reduce to the same kind of further demand that animal organizes in a large amount. Organize materials to send to the relevant laboratories or cells used for others and train within several hours. If has already known a small part of DNA array, can utilize PCR technology to amplify DNA while organizing from few part, enable extremely rare sample ‘ Such as a stick of hair or single sperm) Have higher value and greater credibility.
The organization must be taken from the animal in 1- 2 minutes after the intravital animal or death, and should keep fast, unless the following situations appear: Take a sample in the condition or hasty living subject that arranges of field, so have no time to contact laboratory. Take at this moment? ? ? The destination expands gathering and storing amount. If has already known the precise use of the sample in detail, the condition can be relaxed sometimes. For example, for blood sample, some enzymes do not demand to be frozen and stored immediately.
Every a organization preparation and save method were ranked in tables 7-1, listed the use of each kind of sample in the table at the same time. Freeze the sample while storing and can be put people liquid nitrogen, dry iced or puts directly fast – 70 ¡æ refrigerator. It freeze it can deposit by sample in – liquid nitrogen, refrigerator, dry ices 70 ¡æ or – keep and transport in refrigerators 20 ¡æ. The sample is frozen and stored and transported in order to put the result into liquid nitrogen better. Only if can’t be used to freeze the refrigerator.
1. 2. l Treatment of the soft tissue studied for the chromosome
Soak all or some samples in 0 at room temperature. In 9% physiological saline, send the sample to the laboratory within several minutes, will send within 1- 2 hours too at the latest.
Studying and dyeing physical stamina sees that there is silk splitting and meiosis, thus understand relevant chromosome fall in location. The chromosome will be influenced and fertilized recombinating with heredity while pouring the location, the possible same sex individual should check from chromosome respect, but for mammal, do not usually need to study the other chromosome of same sex.
1. 2. Treatment of 2 organizations used for training as fibrous cells
The tissue that usually comes from the young animal is used in cells and train better. In an individual, it is the best that cancer tissue and lung are organized, but the other soft tissue can be used.
Cell train a commonly used one fresh to organize, but the animal organization several days after catching and killing can still be used for training. In a word, the more the sample quicks,come to the laboratory, the greater the possibility to succeed is.
If need to cut the skin of the intravital animal while taking a sample, should go on under the aseptic condition. To train to cells, does not have
It is very necessary too to carry on further treatment under the fungus condition, but this is different to accomplish in the field. So, a stuffy close space and mask, glove are very useful, the attenbant should leave the mesa of job a little farther at least. All mesa, reagent bottle, glove, instrument,etc. should sterilize with 70% of the alcohol before using. Reagent used should be fresh, it has any suspicion (such as becoming muddy or changing the color) that aseptic to it Should pour out.
Will organize samples to put under the aseptic condition into 0% of the alcohol, after waving for one minute, transfer the sample to people to collect in the liquid, stand at the room temperature for 1- 4 days. If need to stand for a longer time, must organize samples to turn into and transport in the liquid two days later, can preserve for a few days at room temperature. It is very important to operate aseptically in the transfer process, because transport and seldom use preservatives in the liquid.
Collecting liquid and transportation liquid is generally accepted the laboratory of the sample to offer. Can produce more materials to train but needn’t be fetched from the animal again as fibrous cells, get the result of getting twice the result with half the effort.
1. 2. 3 is used in the chromosome the collection of blood specimen studying
Quarrying the blood specimen must be operated aseptically. The syringe used for collecting blood, small bottle,etc. employ Heparin to wet. Have already dealt with Heparin when some articles are bought. Send the blood specimen to the laboratory rapidly ‘ But not make it freeze under the low-temperature condition) . If the condition is not allowed, under the aseptic condition, every 0. Lml the intersection of blood specimen and with 5ml blood specimen deal with liquid, then pass on to get laboratory(laboratory have another kind of prepared the intersection of treatment and liquid) under room temperature .
1. 2. 4 treatment which is used in the enzyme and soft tissue which other proteins study
Cut the soft tissue into small one 1cm square, wrap and freeze and exist rapidly. In benzene formaldehyde substantially as organizing one to soak in 2%, put at the room temperature, the activity of some enzymes can keep for several months (Nakanishi,etc. 1969) . Some fish proteinoid can be analyzed with the material that the market buy, but the purpose here is distinguishing kind, but not in order to get the exhaustive hereditary data.
1. 2. 5 collection which is used in the enzyme and blood specimen which protein studies
It is that test use it employ syringe, test tube,etc. wrap up Heparin ( It had better be lithium salt) ,Or wet with Heparin kept at low temperature. If it is possible, all solutions are formulated etc. and operated and should be finished fast at 5 ¡æ. As to the sample in larger volume ‘ >5ml) ,Had better separate erythrocyte, leucocyte, blood plasma. At odds with the community or the leadership 10 minutes in 1000r/ min at first, draw the blood plasma fast, package and freeze existing rapidly. Then a layer of leucocytes floating on the surface of erythrocyte in aspiration are taken, at odds with the community or the leadership after and then washing the erythrocyte twice with PBS of 5- 10 times of volume, freeze and store the flaky precipitate fast finally.
The leucocyte floor is dissolved with ACK (0. 15mol/ dm3 chlorine ammonium, 0. Hydrogen potassium 01mol/ dm3 carbonic acid, 0. lmol/ dm3 Na2 EDTA) ,Until changing? ? Clarify getting red ‘ About 5 minutes) . In 500r/ min at odds with the community or the leadership 5 minutes later, sucks ACK and erythrocyte chip carefully, the leucocyte left will be suspended in ACK again. 2 minutes later, cells become the group precipitates, washes with PBS at this moment. If the precipitate is still red, repeat the whole course once, and then freeze and store it. Leucocyte ( Best source of enzyme) Also can be through sprawling the blood specimen in equal division in 20% Ficoll, 4. The method in 5% Na2 EDTA, make it subside (10- 15 minutes) ,Use one more bar of suctions and draw the leucocyte from the interface, at odds with the community or the leadership 10 minutes in 1000r/ min, and then freeze and exist. With erythrocyte that Ficoll separate being very much difficult to recover and then.
The blood specimen that some proteins can save by means of others is analyzed: Keep at ¡æ5 and send to the laboratory within several hours; Freeze the whole blood checking ‘ Have not dealt with) ; The whole blood or blood specimen separated dilutes in benzene formaldehyde of volume of 3 times, and keep at room temperature. The electric swimming of protein is a method used for studying the hereditary variety or taxonomy difference in the population. Compared with other vertebrates, the mammalian blood specimen is not very good enzyme and source of protein, fetching the soft tissue sometimes is better.
l. 2. 6 save which is used in the soft tissue that nuclear DNA studies
Will organize cutting small one 1cm square, will wrap up and freeze existing. Or put it into 80% of volume of several times Ethanol( Analyze
Pure) China, preserve at 4 ¡æ. A method is better in two kinds of methods.

l. 2. 7 save which is used in the blood specimen that nuclear DNA studies
The blood specimen can be used and accounted for the blood specimen volume 0. 15% K2 EDTA of 01 times as anticoagulatory pharmaceutical, and can join 0. 05 time protease K ( 0. 1%, the sour grade of core, it is kept that Boehringer Mannheim is icy, ones that uses and unfreezes newly) ,Fully mix, freeze and exist rapidly. Blood and EDTA mixed solution can be put at the room temperature for a few days, but the quality of DNA can not be very good.
DNA technology can analyze any a part of the sample genome of nearly all tissues, relevant problems that thus can overcome and study and make a variation with protein and categorised class hour appears.

1. 2. Save of 8 hair and sperm’s sample used for preparing DNA
Though from drawing DNA drily and in the hair and sperm’s sample fixed with the formalin in the past, but the most reliable save method frozen exists (Impraim,etc. 1987; Thomas,etc. 1990) . One point that must notice is, reduce DNA pollution of the sample of other sources as much as possible, especially the experimenter’s hands, because PCR reacts to micro- DNA it is very sensitive. Better method toast instrument ‘ Centigrade 180 stays for a night) Or the flame is sterilized, other every one things (the glove, test tube, job mesa) used Had better be with a set of new, commercialized products disinfected by ¦Ã ray. Sperm’s sample can be used for regular heredity to analyze, for instance can carry on chain research (Li,etc. 1988) when mating without artificial control too . Certainly, sperm’s sample can also be used for artificial fertilization, it is very important that this is housing.
1. 2. Save of 9 soft tissue and blood specimen used for drawing the mitochondrial DNA
You had better keep the sample at 4 ¡æ, and send to the laboratory as soon as possible. It store blood specimen it is at glucose of having citric acid bag vacuum tube in when,be can if you can’t freeze, deposit on the a few yearsed (preferably on liquid nitrogen) ,But can only keep at the room temperature for several days after unfreezing. Variation and construction pedigree of in geographical distribution in studying the species in analysis of mitochondrial DNAing are especially useful.
1. 2. Save of 10 soft tissue used for offering RNA
RNA will destroy within extremely short time, so must in the intersection of animal and death or live, examine, in the 1- 2 minute rapidly little yuan to cut into 1cm it, it is frozen and existed in the liquid nitrogen and people put.
RNA can be used in the construction of DNA library, it can offer the probe to geneticist, for the analysis of single active gene in individual total DNA. Though the probe coming from other animals can be used, uses the allogeneic probe result to be best.

2. The animal organizes the abstraction of DNA sample
Already there are many methods to draw DNA of the macromolecule quantity at present, we except introduce the regular DNA preparation method in this part, discuss from the micro- sample emphatically even ‘ Such as hair) Draw some new methods of DNA with the outmoded old sample.
2. l Draw the animal total DNA conventional method
Draw the regular step of DNA as follows in the animal organizes:
( 1) Fetch animals and organize about 100mg to organize one to be cut and broken to pieces with the clean scissors in the sample tube.
( 2) Add the following reagent into sample tube:
500¦Ì l STE solution
25 ¦Ì l protease K (Proteinase K, 10 mg/ ml)
75¦Ì l 10% SDS
( 3? ? ? Mix the even trailing and digest 2 at 56 ¡æ Hour above ( Drift along certain time an even once) .
( 4) Add people isopyknic saturation phenol solution, reverse and mix for more than 5 minutes gently (used in the sample that rDNA and RAPD analyze need extraction for 24 hours) .
( 5) 7 000r/ min is at odds with the community or the leadership 5 minutes.
( 6) Take care to move the clear liquid of upper strata in another clean centrifugal tube, add isopyknic phenol and chloroform, and repeat the step ( 4) With ( 5) Extraction course.
( 7) With isopyknic chloroform ‘ Include different fifth alcohol of volume 1/ 25) Repeat the step ( 4) With ( 5) Extraction course.
( Add 3mol/ dm3 NaAc or cold anhydrous ethanol or different propanol of volume of a time of 5mol/ dm3 NH4 Ac, volume of 2 times of volume 1/ 10 in having the clear liquid, in order to precipitate DNA.
( 9) Put – 70 ¡æ sink, settle 2 hour or put and 20 ¡æ sink, settle nights.
( 10) 7 000r/ min is at odds with the community or the leadership 10 minutes, and then wash DNA to precipitate once with 70% cold ethanol. Will precipitate and put in vacuum desiccator or warm case of 37 ¡æ drily.
( 11) With right amount of TE solution (200- 500¦Ì l) Dissolve DNA sample.
( 12) Determine the density ( 260nm) of DNA with the spectrophotometer ,260/ The mere density ratio of 280nm should be here 1. More than 8, otherwise brief on the pollution with protein or phenol.
( 13) Dilute DNA sample into the density of the job liquid needed with TE solution.
( 14) Fetch about 2¦Ì l of samples and measure electric swimming, in order to judge whether DNA degrades and needs to dispel RNA
2. 2 micro- animals organize the preparation method of total DNA for the sample
The method to draw DNA while organizing from the trace was 1989 by Singer-Sam (Walsh,etc. 1991) Develop. The basic principle of this method is to adopt BioRad Chelex 100 (a kind of chela mixture) Draw DNA. This method is simple and convenient, fast, the template that DNA sample can be regarded as PCR directly after drawing. The concrete operating sequence is as follows:
( 1) Fetch a small an icy tissue (less than 1mg, can use and drill through the disinfecting rifle head) ,Or 1- 3 sticks of hair with Mao Gen (need ethanol and sterilized water by 90%, 70% to wash sequentially first, and cut to a hair cadre and divide) ,Or 1- 5¦Ì l blood, join in the centrifugal tube sterilized at high temperature. Add Chelex solution of 500¦Ì l 5% in the centrifugal tube in advance.
( 2) In charge of the sample to stand for an hour or a longer time at 56 ¡æ, until organizing sample into powder (the necessary time of different tissues have nothing in common with each other) .
( 3) Shake for 10- 15 seconds on the oscillator.
( 4) Boil at 95- 100 ¡æ for 15- 40 minutes.
( 5) Shake for 10- 15 seconds on the oscillator.
( 6) In charge of the sample to keep at 4 ¡æ, at odds with the community or the leadership again before reflecting PCR, make Chelex particle precipitate. Fetch, have clear liquid ‘ 1- 10¦Ì l) As DNA template.
2. Preparation method for 3 outmoded, old DNA samples
The research to outmoded, old DNA started in the middle period of the eighties. 1985, Paabo adopt means that clone from ancient for the first time
Draw and determine a section of DNA array in the Egyptian mummy. However, it is much comparatively to widely launch study on old DNA
Gather enzyme chain type polymeriztion (PCR) ( Mullis, Fallona 1987) begun in invention .
Through physics, chemical action of a regular period ‘ From decades to several million years) DNA which doesn’t have often more serious for degradation,hundreds of length of bp bit of DNA only often, and there may be decoration of the base and some and inhibit the material that PCR reacts. So, while drawing DNA from the outmoded or old sample, the most crucial problem is to prevent the pollution of modern DNA. Need to sterilize and deal with all wares, apparatus and reagent that DNA draws operation, drawing the course should go on in ultra net Taizhong. In addition, should have negative contrast when drawing, in order to monitor the pollution that has the other source DNA. It is a comparatively simple and convenient preparation method that now recommend.
( 1) Fetch a little sample ‘ Such as mammalian hide being desirable 1cm small one * 1cm square) ,Deal with the surface of the sample with apparatus such as the scissors, scalpel, remove the top layer of outside pollution of easiest suffering. The hard sample as to skeleton, amber,etc. needs special apparatus of boring with the small steel etc. to obtain samples from inside.
( 2) The surface -treated sample is washed sequentially with 90% ethanol, 70% ethanol and deionized water.
( 3) Add 200¦Ì l deionized water, 10¦Ì l protease K (10mg/ ml) into Eppendorf tube sterilized With (20¦Ì l) of volume 1/ 10 10% ¦Â – sulfhydryl- coloured glaze base ethanol. Sample that deal with put above-mentioned solution, take, cut, break to pieces as much as possible.
( 4) Digest for 48 hours at 56 ¡æ.
( 5) Add isopyknic 5% – 10% Chelex100 solution in the sample tube, shake for 5- 10 seconds on the swirl mixer, then stand for 20- 60 minutes at 98 ¡æ.
( 6) Shake and mix for 5- 10 seconds, and enable its cooling to the room temperature.
( 7) 12 000r/ min has been at odds with the community or the leadership for 5- 10 minutes.
( Take out the clear liquid of upper strata carefully, put and keep in another clean tube.

3 . Abstraction of the plant total DNA
In 1976, Millgan separated the plant DNA by method that Blin and Stafford were described, but these DNA can’t be used for being cloned. After the method of the pollutant in inventing and removing the plant DNA, people can organize the method of DNA to separate the plant DNA by separating animals. It is separated(Bickle,etc. 1977) that the method to remove electrification macromolecule pollutant of plants is nuclear . CTAB (methyl bromide 16 alkyl three ammonium) Separate ( Murray, Thompson 1980) Separate ( Sung, Slightom 1981) with hydrochloric acid / 12 alkyl methyl amimoacetic acid sodium When. In these methods, it is the most extensive that CTAB method is used because of being simple and convenient, fast. Collecting and keeping the method of the plant tissue will have influence greatly on output and quality of DNA. Though can already be from planting successfully
Separate for DNA( Doyle, Dickson 1987) out in thing sample and fossil ,Adopt fresh material, can produce best result, for would rather produce a large number of form, phenol or other secondary the intersection of supersession and kind of product. If the material can’t be drawn at once, should keep in the cold and wet place, for example in the ice chest. If it can necessary to freeze and keep. Without the condition, preferably quick drying (Liston,etc. 1990) in the anhydrous CaSO4 bottle . The abstraction of DNA should go on as soon as possible, in case it degrades ( Pyle, Adams1989) .
There are a lot of factors that can cause DNA to degrade in the course of DNA to draw. It is a physical factor at first. Because DNA molecular weight is relatively large, the mechanical tension or high temperature is very apt to make the DNA rupture. So, should be as light and slow as possible in practical operation, try hard to avoid too many solution shifting and violent vibration,etc., in order to reduce the mechanical tension to the damage of DNA, should avoid the too high temperature at the same time. Secondly, cells endogenous DNA enzymes and cells will cause DNA to degrade while breaking the secondary product released. So in the experiment of drawing DNA, have designed a lot of alternative separation to buffer the liquid, in order to adapt to different plant materials. Separating pH value of buffering the liquid needs improving sometimes, a bit more optimum that pH should avoid being close to degrading enzyme. A bit in 5 more optimum that the great majority degraded enzyme and fat oxygen synthase pH value. 0- 6. Between 0, and DNA enzyme pH value is a bit in 7 more optimum. About 0 ( Dunham, Bryant 1963) . Because under the condition of crossing the acid, DNA takes off the unstability that purine will cause DNA, it is extremely apt to rupture at the place where the base loss, so most plants DNA draw pH value of buffering the liquid it is 8. 0,Some are even 9. 0. Buffer the complex including a large amount of in the liquid, such as EDTA (two amine four second acid of second) , SDS (12 alkyl sulfic acid sodium) , the serum albumin of ox (BSA) ¦Â – the intersection of sulfhydryl- and the intersection of base and ethanol, glutathion, two the intersection of sulphur and the intersection of candy and alcohol (DTT) Su , ascorbic acid (Vc) , polyethylene pyrrole alkane ketone (PVP) , the burnt carbonate (DEPC) of two second bases , bromine second spindle (EB) When (Hallick,etc. 1977) . But because the some above-mentioned materials are cuts enzyme and non- specific nuclease in inhibiting from, so should remove it in the following step. Separate DNA and protein and generally adopt the method that phenol – chloroform collects. Phenol, chloroform has extremely strong denaturation to protein, but not influenced DNA. The following method describes the separation of DNA and pollutant many candies, can also go on with overcast ion-exchange chromatography appearance. Now list the methods of 3 kinds of common separation plants total DNA. 3 kinds of methods dissolve the cell membrane different from method to separate protein, each have its excellent, shortcomings.
3. 1 CTAB buffers the liquid method
This is used for separating the method of the plant DNA widely enoughing, it is applicable to plant most plants, especially when the sample quantity is very small. This law uses the stain-remover CTAB of cation to dissolve the plant cell membrane, and form a complex with DNA. Its advantage does not need to prepare a large number of plant tissues, and is suitable for looking like the leaf, root, seed, embryo, endosperm, pollen and tissue ( Rogers, Bendch 1985) which suspends many types of tissue,etc. trained . In addition, this law expecting much to sample quantity, to the sample (mummy, fossil) of one milligram of quantity while as a child A lot of grams of fresh materials arrives and can be used (Golenberg,etc. 1990; Rogers Bendch 1985) .
3. l. Experiment method of 1 CTAB
1. Material
( 1) 2 times of CTAB buffer the liquid:
100 mmol/ dm3 Tris-HCI is pH 8. 0
1. 4mol/ dm3 NaCI
20 mmol/ dm3 EDTA
2% CTAB( W/ V)
1% PVP-360( W/ V)
0. 2% ? -Sulfhydryl- base ethanol(volume ratio,with before add in being at fume hood)
( 2) Wash and buffer the liquid:
76% ethanol
10 mmol/ dm3 second acid ammonium
( 3) Suspend and buffer the liquid:
10 mmol/ dm3 second acid ammonium
0. 25 mmol/ dm3 EDTA is pH 8.
2. Step
( 1) Heat and separate and buffer the liquid, mortar, pestle to 60 ¡æ.
( 2) People 0 put in the hot mortar. 5- 1. 5g fresh or icy leaf, uses 7. 5ml 2 times of CTAB separates and buffers the liquid to grind (can add some quartz sand help to grind) . Buffer the liquid and grind by a time of CTAB in freezing the organization doing. Quench the organization that is ground into 50ml tube, use 0 more. 5ml 2 times of CTAB buffer the liquid and wash the mortar, will wash the liquid to add to tube.
( 3) Stand 30- 60 minute at 60 ¡æ test tube, then shake gently, make after the its sufficient mixing dropping to the room temperature.
( 4) Add 10ml chloroform – different fifth alcohol (24:1) into test tube Extract liquid ‘ If the color is dark can be carried on once again) ,Jiggling makes it mix, 1500r/ min is at odds with the community or the leadership 5 minutes, enables its point of phases at the room temperature.
( 5) Quench water of upper strata into and 15ml in the tube, add 2/ 3 cold different propanol of volume, mix in order to precipitate sour core gently. If can not see the nuclear acid is precipitated, can stand at – 20 ¡æ for 20 minutes or a longer time.
( 6) At odds with the community or the leadership 3- 5 minute such as 800r/ min under the room temperature, see light group or precipitate, can it stands 20 to be and then at odds with the community or the leadership in minute under 20 ¡æ.
( 7) Pour out, have clear liquid carefully, don’t pour out core to be sour, core at this moment sour to apply bottom that in charge of to fluffy generally, add 15ml, wash, buffer liquid, rotates and washes the precipitate gently, the core will become whiter sourly in 15- 20 minutes.

( At odds with the community or the leadership 5 minute such as 800r/ min (if insufficient, strengthen at odds with the community or the leadership speed or lengthen at odds with the community or the leadership time) at room temperature ,Pour out and wash and buffer the liquid, will be in charge of pouring deducting on the napkin, will let the precipitate be dry, take care not to let DNA slip.
( 9) By suspending and buffering the liquid on a small quantity according to the size of the precipitate ‘ 10- 100 ¦Ì l) Suspend DNA.
( l0) At 37 ¡æ with 100¦Ì g/ ml RNAase Wen breeds for 30- 60 minutes.
( 11) If the organization would rather include only or other secondary products, can further purify (can purify with chloroform, phenol) DNA . Some materials may need to use chlorine cesium (CsCl) Gradient purification. The above-mentioned method can be improved too, the density like sulfhydryl- base ethanol can be improved to 25mmol/ dm3, can also add ascorbic acid of l%, DTT,etc., CTAB can also be replaced (Golenberg,etc. 1990) with SDS ,Can also use TE (10mmol/ dm3 Tris-HCI, pH 8.0, lmmol/ dm3 EDTA) Make and suspend and buffer the liquid.
3. l. 2 CTAB deposition method
1. Material
2 times of CTAB separate and buffer the liquid; Precipitate and buffer the liquid:
100 mmol/ dm3 Tris-HCI is pH 8. 0; 20 mmol/ dm3 EDTA; 2% CTAB( w/ V) .
2. Step
( 1) With 3. 1. The first 4 steps are the same as in 1.
( 2) Draw the water looks of the upper strata, enter its example in a 15ml tube.
( 3) The sediment which adds volume of 3 times buffers the liquid, make the density of NaCI reduce to 0. 35mol/ dm3, stands for 30 minutes at room temperature.
( 4) 2 000r/ min is at odds with the community or the leadership 5 minutes under the room temperature, in order to precipitate DNA.
( 5) According to the size of the precipitate, buffer the liquid (10- 100¦Ì l) by a small amount of suspension Suspend DNA.
( 6) Though it is effective that chloroform or phenol draws DNA, would rather include only the organization of other secondary products to those, can further purify on chlorine cesium gradient.
3. 2 protein precipitates the method
This method can produce high-quality DNA, but the output is the lowest. Generally sink with acid of second potassium before precipitating the nuclear acid
Settle 1983 such as protein and many candies (Dellaporta; Galau,etc. 1988; Hughes, Galau 1988) . This method does not need to be organic to draw, and fast, so is more suitable to a large number of samples.
1. Material
Draw and buffer the liquid: 100mmol/ dm3 Tris-HCI is pH 8. 0; 50mmol/ dm3 EDTA is pH 8. 0; 500mmol/ dm3 NaCl; 2% SDS( w/ v) ; l% PVP-360( w/ v) ; 0. 1% ¦Â – sulfhydryl- basic ethanol ‘ Volume ratio) ,By adding in the fume hood before this.
2. Step
( 1) Grind 1 in the mortar with liquid nitrogen. 0g fresh leaf, can add some quartz sand and help to grind. Store powder finished grinding in – 80 ¡æ refrigerator, before adding and drawing the liquid, don’t let the plant material be melted.
( 2) Add 6ml to draw and buffer the liquid in the icy organization, turn into the centrifugal tube of one 15ml, fully shake for about 30 seconds, make its mixing even, right? ? ? Stand for 20 minutes at 65 ¡æ.
( 3) Add the intersection of 2ml and the intersection of 5mol/ dm3 and the intersection of second and sour potassium (pH 6 in tube. 5),Fully shake evenly, stand for 5 minutes in the frozen water. With the sediment of 12 alkyl sulfic acid that is not dissolved potassium, the majority of protein and many candies are removed as the complex.
( 4) In at odds with the community or the leadership 20 minutes for 4 ¡æ lower 2 000r/ min.
( 5) Aspiration has the clear liquid, quench it into centrifugal tube of a clean 15ml, don’t bring into the particulate matter, then add 4ml different propanol to mix gently, put at – 20 ¡æ.
( 6) Lower at odds with the community or the leadership 15 second such as 2 000r/ min at 4 ¡æ, after DNA precipitate, pour out, have clear liquid, change, deduct 10 minute or a longer time at napkin gently, with the dry precipitate.
( 7) Put the precipitate in 100TE and dissolve, then quench solution into. It is at odds with the community or the leadership in sml no matter in,it is at odds with the community or the leadership minute such as 5,remove by sediment that does not be dissolved.
( Be in charge of the middle and upper clear liquid to quench into another one 1. 5ml in the centrifugal tube, add the intersection of 75¦Ì l and 3mol/ dm3 NaAc (pH 7. 6)With 500¦Ì l cold different propanol, fully mix, stand at – 20 ¡æ for 1- 2 hours, and then transfer at the room temperature for 10 seconds after taking out, make DNA in the tube precipitate.
( 9) Wash, precipitate 10 minute such as thing with the intersection of 500¦Ì l and 80% ethanol, at odds with the community or the leadership 1 minute, dry to precipitate 10 minutes such as thing.
( 10) According to the size of DNA precipitate, use TE (10- 100¦Ì l) of small quantity Dissolve DNA.
3. 3 chlorine cesium method
The principle of this method is according to cesium (CsCl) in chlorine Have and separate the cell composition in different density gradient of China. This method can produce DNA of a large amount of high purity, but time-consuming, and need expensive instrument and equipment, relatively suitable for the material needing the complicated one organically and drawing or precipitating (Carr, Griffith 1987; Weeks,etc. 1986) . In some cases, it may draw the only method of high-quality DNA in the plant containing a large number of secondary products. DNA can purify through a combination in following odd dyestuffs in one balanced the intersection of CsCl and gradient, these dyestuffs are bromine second spindle (Ethidium bromide) , the spindle of Bisbenzimicle, iodination third (propidium iodide) , Bisbenzimide, their different buoyancy density has particular utility in separating different chromosome groups.
3. 4 PCR prepares the law in a small amount
The advantage of this method can purify a lot of samples once, faster, and is smaller than 10mg( 50mm) DNA which leaves fresh can produce enough at material,there are ( Millgan 1992)s usable to lasting experimenting on DNA that be produced . This method existing problem is that DNA precipitate may not be close enough, or can form a close DNA to precipitate perhaps.
1. Material
Separate and buffer the liquid: 200 mmol/ dm3 Tris-HCI is pH 8. 0; 250 mmol/ dm3 NaCI; 25 mmol/ dm3 EDTA;
0. 5 % SDS.
2. Step
( 1) Use 1. 5ml centrifugal tube grinds the small organization sample. Desirable calibre size fresh leaf ‘ < 10mg) ,Freeze in liquid nitrogen
Leaf too available.
( 2) Add the separation of 400¦Ì l to buffer the liquid in the sample that is ground, fasten for 10 seconds with the appropriate whirlpool of speed, open the sample by dispersing.
( 3) At the room temperature 1. The intersection of 5ml and at odds with the community or the leadership 12- 15 minutes such as sample under centrifugal tube, precipitate the inner material of cell.
( 4) Collect and clean the liquid on 300¦Ì l, remove and precipitate.
( 5) Add the intersection of 300¦Ì l and cold different propanol in advance in having clear liquid, move reversely sample 1- 3, enable its sufficient mixing, put at the room temperature at least
Put for 5 minutes.
( 6) The sample in the a little centrifugal tube is at odds with the community or the leadership 20 minutes at room temperature, in order to collect DNA precipitated.
( 7) Remove, have clear liquid, sink, settle 10 minutes at room temperature with 80% of 300¦Ì l’s ethanols.
( At odds with the community or the leadership 12 minute such as sample at room temperature, remove, have clear liquid.
( 9) Wash again with 80% of 300¦Ì l’s ethanols, at odds with the community or the leadership 5 minute at room temperature, remove, have clear liquid.
( 10) At room temperature the dry sample or misses the sample for a night in an hour.
( 11) Suspend samples with 100¦Ì l TE.

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