Chapter 3 E. coli feels the making and conversion of Tai cell
The first upright conduct say
Under the natural condition, a lot of quality grains all can pass a germ coalescence a function to transfer a new host, but generally lack in the artificial set up of the quality grain carry the body this kind transfer the essential mob gene, so can’t complete by oneself to transfer from a cell to another coalescence of cell.If need to carry a quality grain a body to transfer into be subjected to a body germ, need inducement to be subjected to a body germ to produce a kind of brief feeling Tai to take outside source DNA.
The conversion(Transformation) leads the outside source DNA member in to be subjected to body cell and make it acquire a kind of means of new hereditary form, it is a microorganism heredity, member heredity, genetic engineering etc. research realm of basic experiment technique.
Convert what process use is generally been the variation stub that the restriction polishes system blemish by body cell, then don’t contain to slice the mutation body(R ˉ , M ˉ ) that Mao and A Ji turn Mao inside restriction, it can is tolerant of outside source DNA member to get into a body combine stability ground the heredity is to the posterity.Be subjected to body cell through some processings of special methods(like the electric shock method, CaCl2, the RbCl(KCl) equalizes to learn to try a method), the change of palliation happen in the transparence of the cell membrane and become can allow the outside source DNA member get into of feeling Tai cell(Compenent cells).Get into is pass by the DNA member of body cell replication, express the transfer of information of the realization heredity, make is appear by body cell a new hereditary form.Convert through the cell of empress to develop in the sieving the culture media, can immediately sieve a conversion son.(Transformant, then take to have different source DNA member of be subjected to body cell)The in common use feeling Tai cell making method contains CaCl2 and RbCl(KCl) method currently, the RbCl(KCl) legal system have of feeling Tai cell conversion the efficiency be higher, but the CaCl2 methodses be simple to easily go, and its conversion efficiency completely can satisfy generally test of request, make the feeling Tai of cell temporarily need not, can join to share total accumulate 15% of have no germ glycerin in-70 ℃ keep(half year), so CaCl2 methods is usage more extensive.
For the sake of exaltation conversion efficiency, want to consider in the experiment that a few important factors are as follows:
1. The cell grows appearance and density: Don’t use to through many times transfer or keep at 4 ℃ development germ, had better directly transfer germ liquids of used for the making feeling Tai cells from the － 70 ℃ or-20 ℃ germ that the glycerin keep the kind.Cell growth density with just got into a logarithms growth a period is good, can pass monitor to develop a liquid of OD600 to control.The OD600 of the DH5 α germ stub is 0.5:00, cell density at 5 × 107/the ml be or so(the different germ stub circumstance have a dissimilarity), more suitable at this time.Density’s leading Gao or shortage all will influence a conversion efficiency.
2. The quality and density of quality grain: The quality grain DNA which useds for a conversion should mainly be a super and spiral Tai DNA(cccDNA).Convert efficiency and the density of the outside source DNA to become a direct proportion in the certain scope, but when join of the quantity of the outside source DNA be excessive or the physical volume lead greatly, convert the efficiency will lower.The cccDNA with 1 ng can immediately make the feeling Tai cell of 50 μ ls attain saturation.Under the general circumstance, the physical volume of DNA aqua shouldn’t exceed the feeling Tai cell 5% of the physical volume.
3. Try the quality of : Use of try, all need such as CaCl2’s etc.s is the tallest pure degree of(GR.Or AR.), Counteract super and pure water prepare, had better load separately to keep in dry of cold dark place.
4. Prevent°from miscellaneous germ with the pollution of miscellaneous DNA:The whole operation process all should at without under the germ condition carry on, the container use, if leave a heart tube, the tip first had better be new, and Be put out a germ processing by high pressure, allly try to all want to put out germ, and the attention prevent°from a quilt an other trying , DNA Mao or miscellaneous DNA pollute, otherwise all will influence conversion efficiency or turn of miscellaneous DNA to go into, the sieving for ising later, authenticate to bring unnecessary trouble.
This experiment takes the E.coli DH5 a germ stub as to be subjected to body cell, counteract CaCl2 processings, make it be placed in feeling Tai, then and pBS quality grain total heat preservation, the realization convert.Because the pBS quality grain takes the ammonia Xia penicillin anti- sex gene(Ampr), can pass the Amp anti- sex to sieve conversion son.If was subjected to body cell and don’t turn into pBS, the culture media top which then is contain Amp can’t grow.The ability grows on the Amp culture media of is affirm by body cell(conversion son) have already ducted into pBS.Convert the son can withdraw the quality grain of conversion after expand and carry on giving or get an electric shock Yong, Mao to slice etc. authenticate further.
This experiment takes the E.coli DH5 a germ stub as to be subjected to body cell, counteract CaCl2 processings, make it be placed in feeling Tai, then and pBS quality grain total heat preservation, the realization convert.Because the pBS quality grain takes the ammonia Xia penicillin anti- sex gene(Ampr), can pass the Amp anti- sex to sieve conversion son.If was subjected to body cell and don’t turn into pBS, the culture media top which then is contain Amp can’t grow.The ability grows on the Amp culture media of is affirm by body cell(conversion son) have already ducted into pBS.Convert the son can withdraw the quality grain of conversion after expand and carry on giving or get an electric shock Yong, Mao to slice etc. authenticate further.
Section 2 material, equipments and try
A. Material
E. The coli DH5 α germ stub: R ˉ , M ˉ , Amp ˉ ;PBS quality grain DNA: Purchase or the laboratory be self-made, eppendorf tube.
Two. Equipments
The constant temperature shakes a bed and give or get an electric shock hot constant temperature development box, the set type high speed leaves scheming, have no germ work set, low temperature refrigerator, constant temperature water bath pot, ice machine, cent light photometer, little by little move a liquid gun.
Three. Try
The 1. LB solid and liquid culture media:The formula sees chapter 1.
The 2. Amps female liquid:The formula sees chapter 1.
3.Contain Amp LB solid a culture media:will go together with good LB solid culture media high pressure to cool off to 60 ℃ after put out the germ or so, join an Amp storage liquid, make eventually the density spread plank after shaking evenly for the 50 ugs/ml.
4.The wheat Kang Kai culture media(Maconkey Agar):take the 52 g wheat Kang Kai Qiong fat, add the distilled water 1000 mls, the tiny fire boils to the complete deliquescence, the high pressure puts out germ and treat cold to 60 ℃ or so join an Amp storage a liquid to make eventually a density to is a 50 ugs/ml, then shake evenly behind Tu2 Ban3.
The 5.0.05 mols/L CaCl2 aqua:call to take the 0.28 g CaCl2(have no water, analytical pure), dissolve heavy steam water at the 50 mls in, certainly permit to the 100 mls, the high pressure puts out germ.
6.Contain the 0.05 mols/L CaCl2 of 15% glycerin: Call to take the 0.28 g CaCl2(have no water, analyze purely), dissolve heavy steam water at the 50 mls in, join 15 ml glycerin, settle to permit to the 100 mls, the high pressure puts out germ.
Section 3 operates a step
A, be subjected to the development of body germ
Pick lately- activate E from the LB flat panel. The coli DH5 αs single germ fall and inoculate a culture media at the 3-5 ml LB liquid in, 37 ℃ next flap to concuss to develop for around 12 hourses, until the logarithms growth expects behind.Hang that germ a liquid with 1:100-1:50 comparisons inoculate a culture media at the 100 ml LB liquid in, 37 ℃ flaps to concuss to develop for 2-3 hours to the OD 600=0.5 or so.
Two, feel the making of Tai cell( CaCl2 methods)
1, turn the development liquid in to leave a heart tube in, place on the ice for 10 minutes, then leave heart at 4 ℃ next 3000 gses for 10 minutes.
2, leave to up pure, the CaCl2 aquas 10 mls which uses to prepare a cold 0.05 mols/L lightly suspends cell, on the ice place after 15-30 minutes, 4 ℃ next 3000 gses leave heart for 10 minutes.
3, leave to up pure, join the CaCl2 that the 4 mls prepares a cold 0.05 mols/L with 15% glycerin aquas, lightly suspend cell, place on the ice for several minutes, then become a feeling Tai cell to hang a liquid.
4, feeling Tai the cell load separately into the small of 200 μ ls and store to save in-70 ℃ can keep half year.
Three, convert
1, took 200 μ ls to feel that the Tai cell hanged a liquid from-70 ℃ refrigerators, the bottom of the indoor temperature made it defrost and immediately place ice after defrost up.
2, join a DNA aqua(the content be not over the 50 ngs, physical volume not more than 10 μ ls), lightly shake evenly, place the empress of 30 minutes on the ice.
3, 42 ℃ water baths in hot shot 90 or 37 ℃ water bathe for 5 minutes and the hot shot is place in ice to cool off bequickly hind for 3-5 minutes.
4, to join the 1 ml LB liquid culture media(do not contain Amp) in the tube, 37 ℃ flaps to concuss to develop for an hour after mixing evenly and make the germ recover normal growth appearance, and express a quality grain antibiotic anti- sex gene(Ampr) of the coding.
5, take the above-mentioned germ liquid after shake evenly 100 μ l the coating after containing Amp sieving flat panel ascend, right side up placing half an hour, needing a germ liquid drive completely culture media absorb sets upside down to develop Min and 37 ℃ develops for 16-24 hours.
Do 2 to check against in the meantime:
Matched control 1: With together physical volume of have no germ double to steam water to replace DNA aqua, other operation and top homology.Under this normal circumstance should have no germ to fall to appear in containing antibiotic LB flat panel.
Matched control 2: Replace by steaming water with the have no of physical volume germ double DNA aqua, but Tu2 Ban3 take 5 the μ l germ liquid coating in don’t contain antibiotic LB flat panel up, under this normal circumstance should produce a great deal of germ to fall.
Four, compute a conversion rate
Statistics each development Min in of the germ fall a number.
Convert behind in containing antibiotic flat panel long of the germ fall for convert son, fall few calculabilities a conversion son total amount and convert frequency according to this germ in the Min, formula as follows:
Convert sub- total amount=the germ fall the few ×s dilution multiple × conversion to respond the original liquid is total to accumulate/the liquid physical volume of Tu2 Ban3 Jun4
Convert frequency(conversion son number/each DNA)=conversion son total amount/quality grain the DNA join quantity(mg)
Feel Tai cell total amount=matched control 2 germs fall the few ×s dilution multiple × germ a liquid total accumulate/the liquid physical volume of Tu2 Ban3 Jun4
Feel the Tai cell conversion efficiency=conversion son total amount/feeling Tai cell total amount
[Notice] this experimental methods also is applicable to other E.colis to be subjected to body germ the different quality grain of the stub a DNA conversion.But their conversions efficiency combine not necessarily similar.Some conversion efficiency Gao, needing to carry on a conversion liquid many dilution Tu2 Ban3 then can get single germ to fall flat panel, but the some conversion efficiencieses be low, Tu2 Ban3 have to condense(if leave heart) a germ liquid, then can more accurate calculation conversion rate.
Think subject of examination
1. Does the making feel what the principle of Tai cell is?
2. If the matched control originally shouldn’ted grow a flat panel that germ fall up the long in the middle of the experiment some germ fall, you would ever hermeneutic this kind of phenomenon?
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