The conjunction of chapter 5 reorganization quality grain, conversion and sieving
The first upright conduct say
The quality grain has stable credibility and operates a simple advantage.If want a gram of DNA part(<10 kbs) with smaller Long and structure simple, the quality grain wants to compare other any carry bodies to all be good friends with.Carry a body in the quality grain up carry on gram Long, say from the principle that is easy, use first to slice Mao to incise a quality grain DNA and purpose DNA part inside restriction, then the body outside make both connect with each other, use again to gain reorganization quality grain to convert a germ and then complete.But in actually the work, how classify to insert a reorganization quality grain of have the outside source DNA with insert but the oneself wreath turn of carry body member is more difficult.Pass to adjust a conjunction to respond Chinese and Foreign source DNA part and carry the density comparison of body DNA, can carry the oneself wreath of body to turn restriction at certain degree under, can also adopt some special gram Long strategy further, if carry a body to turn to the phosphoric acid etc. come utmost lower oneself wreath of carry the body to turn, can also make use of genetics means,such as α , to with each other repair phenomenon etc. to discriminate reorganization son and don’t reorganize son.
The conjunction reaction strategy which carry a body of outside source DNA part and quality grain includes the following a few kinds:
1, take to have to not and with each other repair outstanding the part carry’s using to slice Mao to carry on digest inside two kinds of different restrictions can produce to take to have to not and with each other repair of glue sex bitter end, this is also the most easy gram the DNA part of the Long, under the general circumstance, in common use quality grain carry a body to all take to have several dissimilarity restriction Mao of identify the sequence constitute of several Long point, as a result almost can always find out the restriction Mao which matches with outside source DNA part bitter end to slice to order of carry a body, thus outside source part definite direction ground gram Long go to carry a body up.Can also when the PCR expand, at DNA part the both ends be artificial to plus different Mao to slice a point in order to with carry a body connect with each other.
2, take to have a glue of homology sex bitter end to use same Mao or together the tail Mao processing can get thus of bitter end. Because the quality grain carries a body and have to also use the same kind of Mao digest, also get 2 of same same alike glue sex bitter end, so is link to respond that Chinese and Foreign source part and quality grain’s carrying body DNA all may take place oneself wreath to turn or a few members***the formation be few to gather a thing, and two kinds of positive and negative conjunction directions may have.So, have to cautiously adjust two kinds of densities of DNAs in the conjunction reaction, in order to make the amount of correct conjunction outcome attain the tallest level.Will also carry body DNA of 5′phosphoric acid Ji the regiment throws away with the alkaline phosphoric acid ester Mao and with maximum limit repress a quality grain DNA oneself wreath to turn.Take 5′carry phosphoric acid of outside source DNA part can availably with go to the phosphoric acid turn of carry a body to connect with each other, produce a takes to have two indentations of open wreath member, is turning into E. The coli is subjected to an expand of the empress of the body germ the indentation in the process and can automatically repair.
3, take to there is even bitter end from produce restriction Mao or nucleic acid of even bitter end to outside slice a Mao digest creation, or is come together Mao by the DNA to repair even with the result that.Because even the conjunction efficiency ratio carry glue sex bitter end to want low have to be many, past in it the conjunction the reaction, T4 DNA conjunction Mao of density and the outside source DNA and carry body DNA, the density all wants high have to be many.Usually still need to join a gather of low density B two Chuns(PEG 8000) to help the DNA member to coagulate gather collective material to convert an efficiency by exaltation.
Special circumstances bottom, outside source DNA the bitter end of the member and use of carry body bitter end can’t match mutually, then can the on-line form quality grain carry body bitter end or the outside source DNA part bitter end to mount to suitablely deal with contact(linker) or link up a head(adapter) to make it match, can also have the usage E of control. The coli DNA comes together the big part part of the klenow of Mao Ⅰ to fill up 3′cave carry, make to match not and mutually of bitter end change for with each other repair bitter end or turn into the even bitter end carries on a conjunction again behind.
What this experiment use’s carrying a physical endowment grain DNA is a pBS and the conversion is subjected to the body germ as E. The coli DH5 α germ stub.Because the pBS up takes Ampr and lacZ gene, the sieving adoption Amp anti- sex of the past reorganization son sieving and α -with each other repair the method that the phenomenon sieving combine together.
Because the pBS takes Ampr gene but outside the top of the source part doesn’t take that gene and the past conversion after be subjected to the body germ only takes conversion son of have the pBS DNA and then can survive down in implying Amp LB flat panel;But the conversion son of outside source part which take to have oneself wreath to turn can’t then survive.This is initial anti- sex sieving.
Take to have β -half the adjust of the lactose Mao Mao gene(lacZ) to control sequence and the β -half lactose Mao Mao N to carry 146 coding sequences of amino acidses on the pBS.This coded to insert a several Long a point in the area, but didn’t break a lacZ reading frame, don’t influence it normal function.E. The coli DH5 α germ stub takes to have β -half the lactose Mao Mao C to carry the coding information of parts of sequences.Under the respectively independent circumstance, pBS and DH5 α code of the β -half lactose Mao the parts of the Mao all have no Mao an activity.But can become protein of have the Mao activity when pBS and DH5 αs merge into an integral whole.The imperfection is near to manipulate the mutation body of gene block on this kind of lacZ gene with take to have an integrity of near manipulate the β -half lactose Mao of gene block sour feminine gender a mutation a body of the realization with each other repairs of phenomenon call α -with each other repair.From α -with each other repair output Lac+ the germ more and easily identifies, it is under distinctive bottom thing X-gal(5-bromine-4 chlorines-3-the Mao Mao-β -D-half lactose Mao) under the existence is induce by the IPTG(different C Ji sulphur generation-the β -D-half lactose Mao) to become blue germ to fall.When the outside source part inserts a pBS quality grain of several Long order the last future reunion to cause to read a code frame change, express egg white to lose to live, the output amino acids part loses α -with each other repair ability, so contain reorganization quality the conversion son of the grain under the same condition at distinctive induce the culture media be last to become white germ to fall.On the wheat Kang Kai culture media, α -with each other repair output Lac+germ because of contain the β -half lactose Mao Mao, can resolve the lactose in the wheat Kang Kai culture media, produce lactic acid, make the pH descend, as a result produce red germ to fall, but after the outside source part insert, lose α -with each other repair ability, as a result don’t produce the β -half lactose Mao Mao, can’t resolve the lactose in the culture media, the germ falls to assume white.Can turn the reorganization quality grain and oneself wreath from here of carry body DNA to separate.This is for the α -with each other repair phenomenon sieving.
Section 2 material, equipments and try
A, material
Outside source DNA part: Make by oneself of take restriction the DNA aqua of the bitter end, the density has been already know; Carry body DNA: PBS quality grain(Ampr, lacZ), withdraw by oneself to purely turn, the density has been already know; Host germ: E. The coli DH5 α , or JM series etc. have α -with each other repair the germ stub of ability.
Two, equipments
The constant temperature shakes a bed, the set type high speed leaves scheming, constant temperature water bath pot, the Qiong fat sugar gel electricity swimwear place and give or get an electric shock hot constant temperature development box, give or get an electric shock a Yong instrument have no germ, work Taiwan, little by little move a liquid gun, eppendorf tube.
Three, try
1, the conjunction respond a buffer liquid(10 ×s):0.5 mols/L Tris ·Cl(pH 7.6), the 100 mols/L MgCl2, 100 mols/L two sulphur Su sugar Chun(DTT)(the percolation put out germ), 500 serum pure egg white of the μ g/ml cows(set cent V.Sigma product)(can use can need not), 10 mols/L ATP.(the percolation put out germ)
2, T4 the DNAs link Mao(the T4 DNA ligase);Purchase finished product.
3, the X-gal keep a liquid(20 mgs/ml): Fuse X-gal to prepare into a keep of 20 mgs/ml a liquid with two A Ji A Mao Ans, wrap with the aluminum screen or the black paper in order to prevent is shine on by light to be break, store on-20 ℃ .
4, the IPTG keep a liquid(200 mgs/ml): Use distilled water to certainly permit to the 1 ml after 800 distilled waters of μ l win fuse a 200 mg IPTG, with 0.22 μ ms filter film percolation in addition to germ, load separately to take care of at the eppendorf and keep in-20 ℃ .
5, the wheat Kang Kai selectivity culture media(Maconkey Agar):Take the 52 g wheat Kang Kai Qiong a fat to add the distilled water 1000 mls, the tiny fire boils to the complete bath a solution, the high pressure puts out germ and treat cold to 60 ℃ or so join an Amp storage a liquid to make eventually a density for the 50 mgs/ml, then shake evenly behind Tu2 Ban3.
6, contain the sieving culture media of X-gal and IPTG:BE in advance making to so contain 50 μ g/ml Amp LB flat panel surfaces to add the 40 ml X-gal to keep liquid and 4 μ lIPTGs to keep a liquid, use to have no germ Bo stick aqua evenly, place in under 37 ℃ place for 3-4 hours, make the liquid complete absorbability of culture media surface.
7, the feeling Tai cell making try: See chapter 3.
8, boil a method to quickly separate a quality grain to try: See chapter 1.
9, quality grain Mao and electricity Yong try: See chapter 2.
Section 3 operates a step
A, the conjunction respond
1, take lately through put out the 0.5 ml eppendorf tube of germ processing, serial number.
2, carry 0.1 μ gs body DNA to transfer to have no germ to leave a heart tube, add etc. Mo Er the outside source DNA of the quantity(slightly many) part.
3, add distilled water to the physical volume is 8 μ ls, re- back by make fire at 45 ℃ heat preservations for 5 minutes of glue to carry solution chain.Will mix a thing to cool off to 0 ℃ .
4, join 10 the × T4 DNA ligase the buffer be one μ l, the T4 DNA ligase is 0.5 μ ls, use to little by little leave scheming to jilt all of the liquid to the tube bottom after mixing evenly, at 16 ℃ heat preservations 8-24 hour.
Do 2 sets to check against reaction in the meantime, among them matched control an only the quality grain carry a body to have no outside source DNA;Matched control two only outside source DNA parts have no quality grain to carry a body.
Two, E. The coli DH5 αs feel the making and conversion of Tai cell
Each conjunction reaction mixs a thing each take 2 μ l conversion Es. The coli DH5 αs feel Tai cell.The concrete method sees chapter 3.
Three, the reorganization quality sieve of grain
1, each conjunction respond that the conversion original liquid takes 100 μ ls to use have no germ Bo stick even coating in sieving culture media up, 37 ℃ descends development half an hour above, until liquid drive complete absorption.
2, set upside down flat panel to continue to develop at 37 ℃ for 12-16 hours, the single germ which treats to appear obviously but doesn’t lap over mutually again fall take out flat panel.
3, put few hour make to show a color at 4 ℃ complete.(this wheat Kang Kai culture media doesn’t do)
Don’t take to there is a cell of DNA, because of having no the Amp anti- sex, can’t in implying an Amp sieving culture media live.Take to there is the conversion son that the pBS carries a body because of having the β -half lactose Mao Mao activity, at the wheat Kang Kai sieving culture media up present to fall for the red germ.Fall for the blue germ at X-gal and ITPG culture media top.Take reorganization quality a grain conversion son because of losing the β -half lactose Mao Mao activity, all fall for the white germ in culture media and x-gal of the wheat Kang Kai selectivity and ITPG culture media top.
Four, the Mao slice to authenticate reorganization quality a grain
Use to have no germ toothpick to pick white single germ to fall to inoculate in contain Amp 50 μ g/mls of in the 5 ml LB liquid culture media, 37 ℃ next flap to concuss to develop for 12 hours.The usage boils a method to quickly separate a quality grain, the DNA directly gives or get an electric shock Yong, in the meantime with boil the method takes out to lift of the pBS quality grain do to check against, there is reorganization quality of insert the part grain electricity Yong to compare with pBS, migration rate slow.Used again and carry with conjunction opposite in response to of slice Mao to carry on Mao to slice an examination further inside restriction.Can also use miscellaneous hand over the method sieving reorganization quality a grain.
[Notice]1, the DNA connect Mao dosage with the property of DNA part relevant, link even together bitter end, have to enlargement Mao quantity, general usage the conjunction glue sex bitter end Mao quantity of 10-10000%.
2, when the conjunction take to have DNA part of glue sex bitter end, the DNA density is generally a 2-10 mgs/ml, at link even together bitter end, need to join a DNA density to the 100-200 mgs/ml.
3, conjunction responds after respond that the liquid stores in 0 ℃ for few day, -80 ℃ store for 2 months, but at-20 ℃ keep conservancy in deep freeze and will lower a conversion efficiency.
4, glue the hydrogen key of sex bitter end formation Be getting more stable under the low temperature, so though T4 DNA conjunction Mao of the most suitable reaction the temperature respond when the conjunction glue sex bitter end for 37 ℃ the temperature takes 10-16 ℃ as to like, even together the bitter end then takes 15-20 ℃ as to like.
5, in the conjunction the reaction, such as wrong carry body member to carry on go to 5′phosphoric acid Ji processing, then use a surfeit of outside source DNA part(2-500%), this will help the oneself wreath that the decrease carries a body to turn and increase outside source DNA and carry the opportunity of body conjunction.
6, the wheat Kang Kai selectivity Qiong fat constitute of flat panel, hold to have conversion son of carry the body DNA to fall for the pale red color germ while imply appropriate antibiotics, but hold have already taken reorganization quality of insert the part a grain a conversion son to fall for the white germ.The effect together blue and white spot sieving of that product sieving, and the price be cheap.But need to pick white germ to fall in time, be development time to prolong, white germ’s fall will become tiny gradually red, the influence choose.
7, X-gal is 5-bromine-4-chlorine-3-the lactose of Mao Mao-b-D-half(5-bromo-4-chloro-3-indolyl-b-D-galactoside) is become with the water solution young man of the half lactose Mao Mao(b-galactosidase) of the derivatives of Mao Mao show blue.IPTG is different C Ji sulphur generation half lactose Mao(Isopropylthiogalactoside), in order to don’t living a rational inducement thing, it can induce a lacZ expression.
8, take conversion son of carry the body DNA to fall, but take reorganization quality of insert the part a grain conversion son to fall for the white germ for the blue germ in implying the sieving culture media of X-gal and IPTG, flat panel such as after 37 ℃ develop is put in refrigerator for 3-4 hours can make to show color reaction well, the blue germ falls obviously.
Think subject of examination
1. Mainly should consider while carrying a body to carry on an outside source DNA part gram Long with the quality grain which factors?
2. Make use of α－ with each other repair phenomenon sieving to take have already inserted part
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