The separation of chapter 1 quality grain DNA, purely turn and authenticate
The first upright conduct say
Pass the reorganization DNA technique to an useful purpose DNA part, send into be subjected to body cell in carry on breed with expression of tool call carry a body(Vector).The germ quality grain reorganizes to in common usely carry a body in the DNA technique.
Quality grain(Plasmid) the stable gene which is the outside of a kind of chromosome, size from 1-200 kbs not etc., for double chain, shut the DNA member of wreath, and exist by super and spiral appearance in host cell.The quality grain is main to discover at the germ and put line germ and fungi cell in, it has an independent replication and transcribes ability, can in the son generation the cell keep Heng settle of copy a number, and express the heredity information take.The replication of quality grain with transcribe and depend on some Maos and protein which code at the host cell, if leave host cell, can’t survive, and the host even have no them and can also normally survive.The existence of the quality grain makes the host have some additional characteristics, such as to anti- sex of the antibiotics etc..F quality grain(call F factor or property grain again), R quality grain(drug-resistant factor) with Col quality grain’s(produce the E. coli plain factor) etc.s is all familiar natural quality grain.
The quality grain generally has 2 in the replication in the cell: close control type(Stringent control) type(Relaxed control) with loose Chi control.The former’s certain stage in the cell period carries on a replication and be chromosome not to make duplicate, it can’t make duplicate, either, usually each inside the cell implies 1 or a few members, like F factor.The quality grain of the latter can make duplicate at any time in the whole cell period and have in each cell many copy, general at 20 above, like Col E1 quality grain.While using protein to synthesize depressant-chloromycetin, protein inside the cell synthesize, replication and cell division of the chromosome DNA all are repress, close type quality grain the replication be stop, but loose Chi type the quality grain continue to make duplicate, the quality grain copy number can from originally more than 20s expand to 1000-3000 and the quality grain DNA shares the content of total DNA and can be increase by original 2% to 40-50% at this time.
The different quality grain which makes use of same replication system can’t exist together in same host cell and be when two kinds of quality grain ducts into same cell in the meantime, they are in the replication and later on the allotment the process of sub- cell each other competition, in some cells, a kind of quality grain gain advantage, but in a little bit another cells another quality grain but gain an advantage.When the cell grows several generation after, share a handful of quality grain will throw to lose, as a result only two kinds of 1 kinds of quality grain in the cell posterity, this kind of phenomenon calls the incompatibility(Incompatibility) of quality grain.But make use of a dissimilarity replication the quality grain of the system can then be coexist with stability ground in same host cell.
Quality grain the gene which usually imply to code some Maos, its form type anti- sex which include to the antibiotics, produce some antibiotics and decline to solve to slice Mao and polish Mao inside complicated organic matter, vegetable and bowel toxin of the creation E. coli and some restrictions etc..
Quality grain carry the body is in the natural quality grain of the top of the foundation carry on for adapting a laboratory operation what artificial set up.Compared with the natural quality grain, quality grain carry a body to usually take to have 1 or more than a selectivity marking gene(like antibiotic anti- sex gene) and an artificial to synthesize of imply and slicing inside several restrictions the Mao identifies a point of several Long point sequence, and threw away greatly part not essential sequence, make the molecular weight reduce possibly, in order to operate at the genetic engineering.Mostly the quality grain carries a body to take some multipurpose assistance sequences, these uses include to authenticate a reorganization gram Long, creation to used for the single chain DNA, body of sequence measurement to outside transcribe an outside source DNA sequence and authenticate through the method naked eye of the organization chemistry part of insert a great deal of expression etc. of direction, outside source gene.An ideal gram Long’s carrying a body mostly should have a little bit following characteristics:(1)The molecular weight is small, much copy, loose Chi control type;(2)The list which has to slice Mao inside the variety in common use restriction slices a point;(3)Can insert bigger and outside source DNA part;(4)Have maneuverable examination form a type.The in common use quality grain carries body size generally between 1-10 kbs, like PBR322, PUC series, PGEM series and pBluescript(brief name pBS) etc..
Separate from the germ quality grain’s DNA methodses all include 3 basic steps: the development germ make the quality grain expand;Collections and crack solve cell;Separate and purely turn a quality grain DNA.The adoption dissolves germ Mao and can break the germ body cell wall and 12 alkyl Huang sour sodium(SDS)s can make the cell membrane crack solve with the Triton X-100.After dissolving germ Mao and SDS or the Triton X-100 processings, the germ chromosome DNA will tie up to round to adhere to on the cell fragment, in the meantime because of germ chromosome the DNA greatly has to be many than the quality grain and easily be subjected to the function of machine dint and nucleic acid Mao etc. but is cut off into different size of line part.When use strong and hot or sour, alkali processing, germ of the line chromosome DNA change sex, but the covalence shut to match a wreath form DNA(the circular DNA of the Covalently closed, brief name cccDNA) two chains and can’t separate mutually, be outside condition instauration normal, the line form chromosome DNA the part be hard to reply sex, but with become sexual protein and cell fragment tie up to round together, but quality grain the DNA double chain recovers original shape again, re- become natural super and spiral member, and exist by fusing appearance at the liquid mutually medium.
In the germ cell, covalence’s shutting wreath quality a grain is exist with super and spiral form.In the process of withdrawing a quality grain in, in addition to super and spiral DNA, will also produce the quality grain of other forms DNA.If there is a chain taking place 1 or deciding crack more in quality grain chains with two DNA, the member can revolve but remove the tension of chain, become the wreath form of loose Chi type member and call to open wreath DNA;(Open circular DNA, brief name ocDNA)If quality grain DNA two chains at same decide crack, then become a line form DNA(Linear DNA).When the quality grain withdraw DNA electricity Yong, same quality grain DNA it the Yong of super and spiral form move speed and compare Yong of open the wreath and line form member to move speed quickly.
Section 2 material, equipments and try
A, material
E with the pBS. αS or the JM series germ stub of the coli DH5, the 1.5 ml plastics leaves a heart tube(call eppendorf a tube again) and leave a heart tube.
Two, equipments
Little by little take a liquid machine(20 μ ls, 200 μ ls, 1000 μ ls), the set type high speed leaves scheming, the constant temperature flaps to concuss to shake a bed, the high pressure steam disinfects a machine(put out a germ pot), the whirlpool flaps to concuss a machine, give or get an electric shock a Yong instrument, the Qiong fat sugar flat panel electricity swimwear place with constant temperature water bath pot etc..
Three, try
1, LB liquid culture media(Luria-Bertani):Call to take the egg white Mao(Tryptone)10 gs, the leaven withdraws the thing(Yeast extract)5 gs, the NaCl 10 gs, dissolve to go to ion water at the 800 mls in, adjust pH to 7.5 with the NaOH, add to ion water to total accumulate 1 rise, the high pressure descends fume to put out germ for 20 minutes.
2, LB solid culture media:Rise to add the 12 g Qiong cosmetics each time in the liquid culture media, the high pressure puts out germ.
3, the ammonia Xia penicillin(Ampicillin, Amp) female liquid:Go together with into a 50 mgs/ml aqueous solution, -20 ℃ keep a back up.
4, dissolve germ Mao aqua: Use a 10 mmols/L Tris ·the Cl(pH 8.0) aqua prepare into a 10 mgs/ml, and load separately into a small to keep after-20 ℃ , each time 1 as soon as using and then to throw .away
5 and 3 mols/l NaAc:(pH 5.2) Fuse a 40.81 g NaAc in 50 ml water ·3 H2 O, adjust pH to 5.2 with the ice acetic acid, fill with water to certainly permit to the 100 mls, the high pressure puts out germ after load separately and store on 4 ℃ refrigerators.
6, aqua Ⅰ :50 mmols/L glucose, 25 mmols/L Tris.Cl(pH 8.0), 10 mmols/L EDTA.(pH 8.0) The aqua Ⅰ can become to criticize to prepare, each 100 mls, the high pressure puts out germ for 15 minutes and store on 4 ℃ refrigerators.
7, aqua Ⅱ :0.2 mols/L NaOH(faced to use the 10 mols/L NaOH female liquid dilution before use), 1% SDSs.
8, aqua Ⅲ :The 5 mols/L KAc 60 mls, the ice acetic acid 11.5 mls, the H2 Os 28.5 mls, certainly permits to the 100 mls, and the high pressure put out germ.Aqua eventually density is: K+3 mols/L, the 5 mols/L of Ac ˉ .
9, the RNA Mao A female liquid:RNA Mao the A dissolve Tris at the 10 mmols/L ·Cl(pH 7.5), go together with into the aqua of 10 mgs/ml in the 15 mmols/L NaCl, heat at 100 ℃ for 15 minutes, make to mix some DNA Maos to lose to live.After cool off with the 1.5 ml eppendorf tube load separately into a small to keep in-20 ℃ .
10, saturated Fen:The city sells to have in the Fen Mao etc. oxide, these outcomes can cause phosphoric acid two split of ester keyses and cause hand over of RNA and DNA allied, should carry on with the congealed tube in 160 ℃ heavy steam.Heavy steam Fen to join 0.1% of 8-the Qian Ji Mao Mao(Be an anti- oxidizing agent), counteract a 0.5 mols/L of wait the physical volume Tris ·Cl(pH 8.0) with 0.1 mols/L Tris ·Cl(pH 8.0) buffer the liquid again and again takes out to lift to make it saturated and make it the pH value attain 7.6 above, because under the acidity condition the DNA will assign in organic mutually.
11, chloroform:press the chloroform:different E Chun=24:1 physical volume ratios join different E Chun.The chloroform can make the egg white change sex also contribute to a liquid mutually with organic mutually of separate, the different E Chun can then rise cancellation to take out to lift to appear in the process of foam. Press the physical volume/physical volume=1:1 hybrid above-mentioned saturated Fen and chloroform namely get Fen/chloroform.(1:1)Fen and chloroform all have very strong causticity, operation should wear gloves.
12, TE buffer liquid:10 mmos/L Tris ·Cl(pH 8.0), 1 mmol/L EDTA.(pH 8.0)The high pressure stores on 4 ℃ refrigerators after put out the germ in.
13, STET:0.1 mols/L NaCl, 10 mmols/L Tris ·Cl(pH 8.0), 10 mmols/L EDTA(pH 8.0), 5% Triton X-100s.
14, STE:0.1 mols/L NaCl, 10 mmols/L Tris ·Cl(pH 8.0), 1 mmol/L EDTA.(pH 8.0)
15, electricity Yong use to try: (1) TBE buffer liquid(5 ×s):Call to take the Tris 54 gs, the boric acid 27.5 gs, and join the 0.5 M EDTA(pH 8.0)20 mls, settle to dissolve to 1000 mls. (2)Last kind buffer liquid(6 ×s):0.25% bromine Fens are blue, 40%(w/v) cane sugar aqueous solution.
Section 3 operates a step
A, the development and collections of germ
Will imply a quality grain a pBS the DH5 α germ kind to inoculate in the LB solid the culture media(contain 50 the μ g/ml Amp), 37 ℃ develops for 12-24 hours.Use to have no germ toothpick to pick single germ to fall to inoculate the 5 ml LB liquid a culture media(contain 50 the μ g/ml Amp) medium, 37 ℃ expects after flap concuss development about 12 hours to the logarithms growth.
Two, quality grain the DNA be a little amount to quickly withdraw
The quality grain quantity with small DNA withdraws a method for make the quality grain that a little amount part purely turns DNA from a great deal of conversion son very useful.These method common characteristics are in great quantities simple and fast, can handle to try kind in the meantime and gain DNA to have must purely degree, can satisfy restriction Mao to incise, electricity the Yong analytical demand.
(A), boil a method
1, develop a 1.5 ml a liquid to pour into an eppendorf tube, 4 ℃ next 12000 ｇss leave heart 30.
2, leave up pure, set upside down a tube several minutes make the liquid flow to the utmost at the toilet paper.
3, precipitate a germ body to suspend in the 120 ml STET aqua, the whirlpool mixs evenly.
4, join a 10 mls lately- prepare dissolve germ Mao aqua(10 mgs/ml), the whirlpool flaps to concuss for 3 seconds.
5, put the eppendorf tube into a boiling water bath, 50 immediately take out behind.
6, use to little by little leave the scheming 4 ℃ next 12000 gses to leave heart for 10 minutes.
7, use to have no germ toothpick to clean germ fragment from the eppendorf tube.
8, take a 20 mls to carry on giving or get an electric shock a Yong check.
[Notice]1. Can pick single germ to fall to be directly carry on boiling a method to withdraw a quality grain DNA from the solid culture media to the E. coli.
2. Boil to add to dissolve germ Mao to there is certain limit in the method, density Gao, germ crack solution the effect be not on the contrary good.Sometimes different dissolve germ Mao can also dissolve germ.
3. Will imply RNA in the quality grain DNA of withdraw, but RNA not the interference tests further, such as restriction inside slice Mao digest, second gram Long and conjunction reaction etc..
(Two), alkali method
1, take the 1.5 ml development liquid to pour into the 1.5 ml eppendorf tube, 4 ℃ next 12000 gses leave heart 30.
2, leave up pure, set upside down a tube few minute make the liquid flow to the utmost at the toilet paper.
3, the germ body precipitate heavy suspend in 100 μ l aqua Ⅰs(need violent flap to concuss), the bottom of the indoor temperature places for 5-10 minutes.
4, join lately- prepare aqua Ⅱ 200 μ ls, cover a tight tube, quickly gentle reverse eppendorf to take care of several times to mix evenly a contents thing(never flap to concuss), the ice bathes for 5 minutes.
5, join 150 μ ls to prepare cold aqua Ⅲ , cover a tight tube, and set upside down to leave a heart tube, the geniality flaps to concuss 10, make to precipitate to mix evenly, in the ice bath 5-10 minutes, 4 ℃ next 12000 gses leave heart for 5-10 minutes.
6, ascend the pure liquid move to go into clean join Fen/chloroform(1:1) of wait the physical volume in the eppendorf tube, flap to concuss to mix evenly, 4 ℃ next 12000 gses leave heart for 5 minutes.
7, move water mutually goes into clean join 200% physical volumes in the eppendorf tube of have no water ether, flap to concuss to mix evenly is postpose in-20 minutes in 20 ℃ refrigerators, then 4 ℃ next 12000 gses leave heart for 10 minutes.
8, leave up pure, open a tube to set upside down to make at the toilet paper all liquid run offs, join a 1 ml 70% ether to wash to precipitate once, 4 ℃ next 12000 gses leave heart for 5-10 minutes.
9, absorb in addition to ascend pure liquid, set upside down a tube at the toilet paper to make the liquid flow to the utmost, the vacuum dry 10 minutes or indoor temperature are dry.
10, will precipitate to dissolve in 20 the μ l TE buffer the liquid(pH 8.0, contain 20 the μ g/ml RNaseA) be medium and keep in-20 ℃ refrigerators in.
[Notice]1. Withdraw the process should as far as possible keep low temperature.
2. Withdraw a quality grain to remove egg white in the DNA process very important, adopt Fen/chloroform to clean egg white effect to compare alone use Fen or chloroform like, egg white as far as possible in addition to clean need many times take out to lift.
3. Precipitate DNA to usually use ice ether, place time under the low temperature condition slightly long can make the DNA precipitate completely.Precipitate the DNA can also use different C Chun(general usage etc. physical volume), and precipitate completely, speed quick, but often precipitate salt down, so the most still keeps using ether.
(Three), the Wizard a little amount DNA purely turns system
The Wizard a little amount DNA of Promega company purely turns system and can quickly and effectively take out to lift quality grain DNA and the whole process needs 15 minutes.The quality grain of withdraw can directly used for DNA to measure a preface, Mao to slice analysis and body to outside transcribe etc..
In that system containing to try and post can used for 50 separations which develop a liquid with purely turn, try to include a 10 ml cell to suspend a liquid, the 10 ml cell crack solution liquid;The 10 mls moderates a liquid, the 50 ml Wizard a little amount DNA purely turns resin, and the 50 ml pillar washes liquid(the usage added 95% ether to the 120 mls ago) and 50 Wizard miniature pillar.
The 1 and 1-3 mls stays overnight the liquid 4 ℃ next 12000 gses of the development cell to leave heart for 1-2 minutes.
2, clean up the pure liquid, the germ body cell suspends to suspend a liquid at 200 cells of μ l in, well hybrid, and move into an eppendorf tube.
3, add 200 μ l cell crack solution liquids, reverse to leave heart to take care of several times, until the aqua become clear.
4, add 200 μ ls to moderate a liquid, reverse to leave heart to take care of several times.
5, 4 ℃ next 12000 gses leave heart for 5 minutes and take up the pure liquid in new eppendorf tube.
6, add the 1 ml Wizard a little amount DNA to purely turn resin, reverse to leave heart to take care of several times to well mix evenly.
7, take 1 time to inject a machine, take out to note to fill, and make to inject tube and the Wizard miniature pillar conjunction, use to move a liquid gun will above-mentioned mixture the liquid join to inject a tube in, counteract to note to fill to lightly push, make the mixture get into miniature pillar.
8, will inject machine and miniature pillar to separate, take out to note to fill, will inject tube and miniature pillar again connect with each other, join a 2 ml pillar to wash a liquid, counteract to note to fill to lightly push, make the pillar wash a liquid into miniature pillar.
9, take out miniature pillar to place in eppendorf tube, leave heart for 2 minutes to remove miniature pillar in of the pillar wash a liquid.
10, put miniature pillar in a new eppendorf tube, add 50 μ l TE(or water) after miniature pillar win, holding still for a minute, 4 ℃ next 12000 gses leave heart 20.
11, throw miniature pillar away, take care of eppendorf medium quality grain DNA to store at 4 ℃ or-20 ℃ refrigerators.
[Notice] resin usage should ago well mix evenly, if have a crystallize, can use resin 25-37 ℃ water baths handle for 10 minutes.
Three, quality grain the DNA in great quantities withdraws and purely turns
Demand in the creation the Mao table, the measurement the sequence, the making the probe etc. the experiment Gao Chun2 Du4, high density’s quality grain DNA, need to in great quantities withdraw a quality grain DNA for this.The quality grain in great quantities withdrawn the DNA generally needs to further and purely turn and in common use pillar layer Xi method and the chlorination unique steps degree leave a heart method.
(A), alkali method
1, take to develop to the logarithms growth to expect behind of contain the development liquid 250 mls of the germ of pBS quality grain, 4 ℃ next 5000 gses leave heart for 15 minutes and leave up pure, will leave a heart tube to set upside down to make to flow to the utmost up all of the pure liquid.
2, precipitate a germ afresh is suspend in a 50 mls with the ice prepare cold STE in.(this can abridge)
3, synchronously and suddenly 1 methods leave heart to collect germ cell.
4, is suspend in germ sediments afresh the 5 ml aqua I in, well suspend a germ body cell.
5, join the 12 mls lately- prepare aqua II, cover a tight bottle a cover, slowly reverse to leave heart to take care of several times to well mix evenly a contents thing, the ice bathes for 10 minutes.
6, add a 9 mls to prepare cold aqua III with the ice, wave to leave heart to take care of several times to mix evenly a contents thing, place on the ice for 15 minutes, should become a white Xu form to precipitate at this time.
7, 4 ℃ next 5000 gses leave heart for 15 minutes.
8, take up the pure liquid, join the 50 ml RNA Mao A(10 mgs/ml), 37 ℃ water bathe for 20 minutes.
9, join etc. the saturated Fen/chloroform of the physical volume, flap to concuss to mix evenly, 4 ℃ next 12000 gses leave heart for 10 minutes.
10, take upper level water mutually, join etc. physical volume chloroform, flap to concuss to mix evenly, 4 ℃ next 12000 gses leave heart for 10 minutes.
11, take upper level water mutually, join 1/the 4 mols/L NaCl of 5 physical volumes places on the ice for 60 minutes with 10% PEGs(molecular weight 6000).
12, 4 ℃ next 12000 gses leave heart for 15 minutes, precipitate to wash away dirt with the few mls 70% icy cold ether and 4 ℃ next 12000 gses leave heart for 5 minutes.
13, the vacuum take out stem to precipitate and dissolve in the 500 ml TE or water.
[Notice]1. Withdraw process in should as far as possible keep low temperature.
2. After joining aqua II and aqua III operate should mix, slice to hate violent flap to concuss.
3. In order to often existing DNA Mao in the RNA Mao A, make use of the RNA Mao ovenware characteristic, should first carry on a hot processing(80 ℃ an hour) to the Mao’s liquid while use, make the DNA Mao lose to live.
(Two),Wazard a great deal of DNA purely turns system
The alkali method in great quantities withdraws DNA to usually need very long time.The Wiazrd of the Promega company a great deal of DNA purely turns system since simple and fast, needs to leave heart and vacuum to take out stem, this system can from the 500 ml the development the liquid in 3 hours in acquire the quality grain of the 1 mg above high quality DNA.(200-20000 bps)That system doesn’t need Fen and chloroform to take out to lift and purely DNA after turn’s dissolve don’t in water or TE buffer liquid and not contain any salt, can directly used for the DNA sequence analysis and Mao to slice reaction, can be also used for carrying on a body to outside transcribe reaction under the nucleic acid Mao depressant(like RNasin) existent condition etc..
Have in that system of try and post can used for 10 separations which develop a liquid with purely turn, try to include: The 150 ml cell suspends a liquid, the 150 ml cell crack solution liquid, the 150 mls moderates a liquid, 100 ml Wizard a great deal of DNA purely turns resin, the 125 ml Wizard post washes to take off aqua and 10 Wizards to take to have saving leave the post of heart tube.
The cell development of the 1 and 100-500 ml liquid places to leave a heart tube in, 22-25 ℃ next 5000 gses leave heart for 10 minutes and gain cell to precipitate well is suspend in cell to suspend a liquid in.
2, add a 15 ml cell crack a solution aqua combine lightly hybrid, can again and again set upside down a mixture, but can’t flap to concuss with the whirlpool, cell crack solution complete, the aqua will become pure, this one step need 20 minutes.
3, add a 15 mls to moderate aqua, immediately and again and again set upside down to leave heart to take care of several times, and make it mix evenly.
4 and 14000 gs, 22-25 ℃ leaves heart for 15 minutes.
5, carefully will ascend a pure liquid to suck out and move to 1 lately leaves a heart tube in.
6, add the different C Chun of 0.500% physical volumes, hybrid and even, 14000 gs 22-25 ℃ next leave heart for 15 minutes.
7, leave up pure, suspend DNA to precipitate a liquid at the 2 ml TE buffer in.Perhaps have in this one step of precipitate indissoluble.
8, add a 10 ml Wizard a great deal of DNA purely turns resin aqua, and whirlpool mixture.
9, each sample, use a post with a great deal of Wizard, the head of post puts on the vacuum machine.(Promega product, and this kit)
10, turn the resin/DNA hybrid liquid into a post, the vacuum samples the resin/DNA mixture liquid.
11, add a 13 ml a post to wash to take off aqua after taking out the resin/DNA hybrid liquid stem to leave a heart tube in, to tube bottom of the resin/DNA carry on washing to take off(the post is revolve and joining to wash to take off a liquid), and join a post.
12, the vacuum take out what stem join to wash to take off.
13, add a 12 ml a post again to wash to take off a liquid to enter a post and take out stem.
14, join a 5 mls 80% the ether Piao wash pillar in of resin, the post vacuum puts the post into a customer to provide after take out the stem of leave a heart tube in, the 2500 rpms(1300 gs) leaves heart for 5 minutes.
15, take out a post, the vacuum takes out to do for 5 minutes and put the post into what system provide again to leave a heart tube in, the 2500 rpms(1300 gs) leaves heart for 5 minutes.
16, join a 1.5 mls 65-70 ℃ to prepare in the post hot lead of put out germ heavy steam water or TE, the 2500 rpms(1300 gs) leaves a heart post after a minute/leave heart to take care of for 5 minutes.
17, take out a post, leave the aqua in the heart tube for the quality grain for withdraw DNA, can directly put in leave the heart tube, cover cover, the storage is in 4 ℃ or-20 ℃ back up.
[Notice]1. Before use, the post provide by system washes to take off a liquid to press 1:1 join a 125 mls 95% ether.
2. Purely turning resin after hasing to mix evenly use again.
(Three),The Sephrose 2 B pillar purely turns a quality grain DNA
The quality grain that the alkali method withdraw DNA even uses a RNA Mao processing and still imply a little amount RNA.When some DNA experimented to need without the RNA pollution product, then need to be carry on to further and purely turn.Generally in common use Sepharose 2 Bs or Sepharose 4 Bs carry on purely turning, that method has quickly, condition geniality, repeated good, carry the body material can make use of etc. advantage again, as a result have already extensively used for a quality grain, the DNA purely turns.
1, Sepharose 2 Bs last pillar after containing the TE(pH 8.0) balance of 0.1% SDSs.
2, will at most the DNA aqua with 1 ml spread on the Sepharase 2 B pillar.
3, need the upper part conjunction that the DNA aqua completely gets into an empress inside the pillar immediately in the pillar to imply 0.1% TEs(pH 8.0) of SDSs to store a liquid bottle.
4, take the 1 ml run off liquid as 1 to carry on collections.
5, measurese its OD260 values to each tube, with assurance which tubes in imply a quality grain DNA.Usually the quality grain DNA is in the pillar the top the first Feng of run off.
6, the merger is all contain a quality grain of wash and take off liquid, use etc. physical volume of the Fen/chloroforms(1:1) take out to lift and 4 ℃ next 12000 gses leave heart for 2 minutes and turn into upper level water mutually a new tube.
7, join 200% physical volumes of icy cold have no water ether, -20 ℃ next precipitate for 10 minutes, then 4 ℃ next 12000 gses leave heart for 10 minutes and leave to ascend pure liquid.
8, precipitate to add 70% ether rinses, 4 ℃ next 12000 gses leave heart for 10 minutes and leave to ascend pure liquid.
9, precipitate vacuum to take out stem, is dissolve in TE or have no germ water afresh in.
[Notice] in the process of packing pillar in, prevent°from to appear to split or annoy bubble phenomenon in the pillar bed, make the interface keep neat.Contain 0.1% TE balances of SDSs to make the gel in the pillar even towards lately packing become of pillar, apply.
Think subject of examination
1. What basic property does quality grain have?
2. Which improvements does the quality grain carry a body to compare with natural quality grain to have?
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