Chapter 4 RNA withdraws to synthesize with cDNA
The first upright conduct say
Withdraw mRNA from truely the pit the living creature of organization or cell, converse to record to synthesize cDNA through the reaction of Mao Cu the first chain and the second chain, double chain cDNA with carry a body conjunction, then convert to expand, can immediately acquire a cDNA library, set up of the cDNA library can used for true pit living creature gene of structure, expression with adjust the analysis for control;Compare the cDNA can make sure that containing sub- existence and understanding processes to wait a series of problem after transcribe with corresponding a gene set a DNA sequence difference.In fine the cDNA synthesize to have become to really check the basic means of molecular biology nowadays with gram Long.From 70’s middle period head the example cDNA gram the Long publish, have already developped many methods that the exaltation cDNAs synthesize an efficiency, and consumedly improved to carry system to govern, cDNA’s synthesizing to try have already commercialize currently.The cDNA synthesize and gram the basic step of the Long include with the reversal record Mao to synthesize cDNA the first chain, come together Mao to synthesize cDNA the second chain, join to synthesize to deal with contact and double the chain DNA gram the Long arrive to carry a body at the adequacy.(bite germ body or quality grain)
A, the RNA make
The quality of template mRNA directly influences the cDNA synthesize of efficiency.Because mRNA member of the structure characteristics be easily respond by the attack of RNA Mao but decline a solution, plus RNA Mao extremely stable and extensive existence, as a result in the process of withdraw in want strictly a pollution of prevent°from the RNA Mao, and try to repress it activity, this be the key of this experiment success or failure.All exist RNA Mao in all organizations, the person’s skin, finger, try , container…etc. to be probably polluted all, so all need to wear gloves to operate in all experiment processes and usually replace.(use a time of gloves)The glassware use needs to be place to bake more than 2 hour at 200 ℃ in the dry oven.Every can’t use the material of heat baking all can use 0.1% burnt carbonic acids two B ester(DEPC) aqueous solutions a processing such as plastics container’s etc., again use distilled water blunt clean.The DEPC is the chemistry modifier of the RNA Mao, it set with the live Ji regiment of the RNA Mao the wreath of Mi Shi with sour ammonia respond but repress Mao an activity.DEPC and ammonia aqueous solution’s mix will produce a carcinogen, as a result usage need to be be careful.Experiment can also use a DEPC processing using to try, join DEPC to 0.1% densities, then violent flap to concuss for 10 minutes, boil 15 minutes or high pressure again to put out germ to remove survive DEPC, otherwise DEPC also ability and adenine function but break mRNA activity.But DEPC ability and An and Liu Ji respond, as a result contain try of Tris and DTT can’t use a DEPC processing.The Tris aqua can prepare with water of DEPC processing then the high pressure put out germ.Aqua of prepare such as can’t the high pressure put out germ, can use a DEPC processing water to prepare, and use possibly in try of Kaifeng never.In addition to DEPC, can also use different sulphur cyanic acid Gua, the Fan oxygen pit Mao sour and compound, the RNA Mao repress egg white etc..In addition, in order not to mRNA or cDNA adsorb on the glass or the plastics container tube wall, all containers all need Be turned a processing by Huo alkane.
The total RNA’s making’s method is a lot of inside cell, such as different sulphur cyanic acid Gua hot benzene Fen method etc..Many companies have ready-made total RNA to withdraw to try a box, can quickly and availably withdraw the total RNA of high quality.The total RNA of separate can make use of mRNA 3′the bitter end imply to much gather(A)+ of characteristics, be RNA to flow through oligo(dT) cellulose pillar, under high salt buffer liquid function, the mRNA is differently adsorbed especially on the oligo(dT) cellulose, then lowers salinity to wash to take off gradually, in the low salt aqua or the distilled water, the mRNA is wash under.Pillar through two oligo(dT) cellulose can get more pure mRNA.The mRNA purely turned is in 70% ether-70 ℃ can keep above for a year.
Two, cDNA the first chain synthesize
All methods which synthesize cDNA the first chain want to use to depend on to come together Mao(the reversal record Mao) at the RNA DNAses the catalyst respond.The virus(MLV) reversal of the rat leukemia which commercializes reversal to record Mao to have to separate from birds become birds that the Sui cell lump virus purely turns to become Sui cell virus(AMV) to converse to record the E. coli that the leukemia virus of the Moloney rat reversal that the Mao turns with Long from the expression gram records Mao gene currently records Mao.The AMV reversal records Mao to include two many Jis with second Maos that how many grows a Mao activity, these activities include to depend on to synthesize at the RNA DNA, the DNA which depends on in the DNA synthesize and to the DNA:the RNA be miscellaneous to hand over the RNA part of body to carry on inside slice to decline a solution.(RNA Mao H activity)The MLV reversal records Mao to only have single many Jis with second Maos, and fully is depends on in RNA and depend on to synthesize an activity at the DNA DNA, but declines solution RNANA miscellaneous hand over the ability of the RNA in the body weaker, and to hot of stability compare the AMV reversal record Mao bad.The MLV reversal records Mao and can synthesize longer cDNA.(such as big in 2-3 kbs)The AMV reversal records Mao and MLV reversal to record Mao to make use of the most suitable pH value for RNA template to synthesize cDNA, the most suitable salinity doesn’t same alike be each with the most suitable glasshouse, so synthesize the first chain correspond an adjustment condition is count for much.
AMV reversal’s recording Mao and MLV reversal to record Maos has to have already led a thing to come to synthesize of start DNA.The cDNA synthesizes to most in common usely lead a thing is with true pit cell mRNA member 3′carry poly(A) to combine of 12-18 pit the Mao be sour long of oligo(dT).
Three, cDNA the second chain synthesize
CDNA the second chain synthesize a method to have the following a few kinds:
(1) oneself leading the method synthesize of single chain cDNA 3′carry can become one short hairpin structure, this for the synthesize of the second chain provided to ready-madely lead a thing, be the first chain after synthesize the DNA of respond the outcome: the RNA is miscellaneous to hand over a chain to change sex make use of E. coli DNA to come together the Mao Ⅰ Klenow part or reversal to record Mao to synthesize cDNA the second chain, the S1 nucleic acid Mao which finally uses to the single chain particularity digests that wreath, then further gram Long.But the oneself leading synthesize the method more difficult control to respond, and use none exception for the S1 nucleic acid Mao to incise hairpin structure ground and will cause rightness should at the mRNA 5′carry a sequence to appear imperfection and heavy row, as a result that method seldom uses currently.
(2) the displacement synthesize method’s that method to make use of the first chain is in the reversal record output cDNA under the Mao function:the mRNA be miscellaneous to hand over need not alkali of chain to change sex, but exist in the dNTP under, make use of RNA Mao H at miscellaneous hand over the mRNA chain of chain up result in to slice and indentation.Produce a series of RNA to lead a thing thus, make it become synthesize a lead of the second chain a thing, in the E. coli DNA come together the function bottom of Mao Ⅰ to synthesize the second chain.Should respond have 3 main advantages: (1) Very valid; (2) Direct make use of the first chain respond an outcome, need not a further processing and purely turn; (3) Need not use the S1 nucleic acid Mao to incise a pair of chains cDNA in of single chain hairpin wreath.Synthesize cDNA often currently to adopt that method.
Four, cDNA member gram Long
Have already made a good double chains cDNA with general DNA similar, can insert a quality grain or bite a germ body in, is this, essential first link up deal with contact(Linker), deal with contact can be limit inside sex slice Mao to identify a point part, can also make use of bitter end to transfer Mao is carry the bitter end of body and double chain cDNA to mount one few gather dG and dC or dT and dA tail, after backing fire become reorganization quality grain, and convert to be in the host germ carry on expand.Synthetic cDNA can also after PCR expand again gram the Long go into adequacy to carry a body.
Section 2 moves plant organization mRNA to withdraw
A, material
The paddy rice leaf’s slice or small rat liver organize.
Two, equipments
Grind Bo, freeze a set type high speed to leave scheming, low temperature refrigerator, freeze vacuum desiccator, purple outside examination instrument, give or get an electric shock a Yong instrument, give or get an electric shock Yong slot.
Three, try
1, have no RNA the Mao put out germ water:Use the heat bake of the glass bottle(180 ℃ 2 hour) pack distilled water, then join 0.01% DEPCs(physical volume/physical volume) and the high pressure puts out germ after handling to stay overnight.
2, 75% ether:Use a DEPC processing water to prepare 75% ether,(put out germ container to prepare with the heat) , then pack the glass bottle which bakes into the heat in, deposit at the low temperature refrigerator.
3 and 1 Xi pillars add a kind buffer a liquid;20 mmols/L Tris ·Cl(pH 7.6), 0.5 mols/L NaCl, 1 mmol/L EDTA(pH 8.0), 0.1% SDSs.
4, wash to take off a buffer liquid:10 mmols/L Tris ·Cl(pH 7.6), 1 mmol/L EDTA(pH 8.0), 0.05% SDSs.
Four, operate a step
(A)move the plant total RNA to withdraw-Trizol method
The Trizol method is applicable to organization or development germ of mankind, animal, plant, microorganism and the sample measures from several 10 milligrams to a few grams.Use the total RNA that the Trizol method withdraw in no way egg white and DNA to pollute.The RNA can directly used for Northern spot analysis, the spot is miscellaneous to hand over, Poly(A)+separate, the body outside translates and the RNase seals Zu analysis and member gram Long.
1, join the 1 ml Trizol liquid to grind by 50-100 mg organization again after the liquid whet into powder in the N the organization, notice sample is total to accumulate 10% that can’t exceed the Trizol physical volume use.
2, grind liquid indoor temperature to place for 5 minutes, then join the comparison with 0.2 mls to join chloroform by each 1 mlTrizol liquid, the cover tightly leaves a heart tube, by hand violent shake to concuss to leave heart to take care of 15.
3, take upper level water mutually in on lately leaving a heart tube, the comparison which presses the each mlTrizol liquid to add the 0.5 mls different C Chun joins different C Chun, the indoor temperature places for 10 minutes, and the 12000 gs leaves heart for 10 minutes.
4, leave to ascend pure liquid, the comparison which presses each liquid of ml Trizol to join at least a 1 ml joins 75% ether, the whirlpool mixs evenly, and 4 ℃ next 7500 gses leave heart for 5 minutes.
5, be careful to leave to ascend pure liquid, then indoor temperature or vacuum dry 5-10 minutes, the attention doesn’t want the aridity excessively otherwise lowers a RNA deliquescence degree.Then dissolve RNA in water, when it’s necessary can 55 ℃ -60 ℃ water dissolve for 10 minutes.The RNA can carry on mRNA separation, or store to save at 70% ether and keep in-70 ℃ .
[Notice]1, the whole operation want to take mouth-muffle and a time of gloves, and operate under the low temperature possibly.
2, before adding chloroform of evenly strach a liquid can at-70 ℃ keep for more than a month, RNA’s precipitating in 70% ether can keep in 4 ℃ for a week, -20 ℃ keep for a year.
(Two)the mRNA withdraw
Because the mRNA bitter end implies many polies(A)+, be total RNA to flow path oligo(dT) cellulose, under high salt buffer liquid function, the mRNA is especially differently adsorbed pillar at the oligo(dT) cellulose up, in the low salinity or the distilled water, the mRNA can be wash under, through two oligo(dT) cellulose pillar, can get more pure mRNA.
1, use a 0.1 mols/L NaOH to suspend the oligo(dT) cellulose of the 0.5-1.0 g.
2, will suspend a liquid to pack to go into a time of layer Xi pillar of put out the germ or pack go into fill to have through DEPC processing and put out the glass cotton of the germ through the high pressure of in the straw of Ba Si De2, the pillar bed physical volume flushes a pillar bed with 300% put out of pillar bed physical volumeses germ water for the 0.5-1.0 mls.
3, use the 1 x pillar layer Xi to add a kind buffer a liquid to flush a pillar bed, until the pH value of run off liquid is small in 8.0.
4, will(a) medium the RNA liquid withdraw after 65 ℃s teach 5 minutes and quickly cools off to the indoor temperature and join etc. the physical volume 2 x pillar layer Xi buffer liquid, last kind, immediately use to put out a germ tube collections to wash a liquid, be all RNA aquas to after get into the pillar bed join the 1 x of 100% pillar bed physical volumes a layer Xi pillar to add kind aqua.
5, measurement each a tube of OD260, be wash the OD in a liquid is 0:00, join 2-300% put out of pillar bed physical volumes the germ wash to take off a buffer liquid, with 1/3-1/2 pillar bed physical volume cent take care of collections to wash to take off a liquid.
6, the measurement OD260, the merger implies wash of RNA to take off for a set of cent.
7, join 1/the 3 M of 10 physical volumes NaAc(pH 5.2), the icy cold ether of 2.500% physical volumes, mix evenly, -20 ℃ be 30 minutes.
8, 4 ℃ next 12000 gses leave heart for 15 minutes and carefully leave to ascend pure liquid, wash Di to precipitate with 70% ether, 4 ℃ next 12000 gses leave heart for 5 minutes.
9, be careful to leave to ascend pure liquid, precipitate air for dry 10 minutes, or the vacuum be dry 10 minutes.
10, fuse a RNA liquid with a little amount water, can immediately used for cDNA to synthesize.(or keep in 70% ether to also store to save in-70 ℃ )
[Notice]1, the mRNA is in 70% ether-70 ℃ can keep above for a year.
2, oligo(dT) cellulose the pillar can use a 0.3 mols/l NaOH to wash clearly, then add the kind buffer liquid balance with the 1 F Xi pillar after use, and join 0.02% to fold a nitrogen sodium(NaN3) refrigerator conservancy, repeat an usage.Need to add a kind buffer with a Xi pillar before use each time, the liquid one by one in order pours to wash a pillar bed.
The section 3 plant virus RNA withdraw
Mostly the plant virus RNA is a single chain RNA, and it pole is homology to the mRNA pole, the plant virus RNA withdraws more and in brief, general use Fen chloroform can immediately acquire a satisfied result.
A, material
Lift pure TMV virus a liquid(10 mgs/ml).
Two, equipments
The frozen set type leaves scheming, the low temperature vacuum dry instrument, electricity Yong instrument, give or get an electric shock Yong slot.
Three, try
TE-saturated Fen:chloroform(1:1), chloroform, the 3 M NaAc(pH 5.2), ether(100% and 70%), TE buffer liquid, have no RNA Mao of double germ water.
Four, operate a step
1, take an eppendorf tube to join to lift a pure TMV(10 mgs/ml)400 mls, again join etc. the physical volume Fen/chloroform, cover the tight tube cover by hand and well flaps to concuss behind for a minute and 4 ℃ next 12000 gses leave heart for 10 minutes.
2, absorb water mutually at one new eppendorf tube, again take out to lift with the Fen/chloroform, until water mutually hand over interface with organically mutually have no egg white.
3, absorb water mutually a tube at the new eppendorf heart, join etc. physical volume chloroform, by hand set upside down to leave heart to take care of several, 4 ℃ next 12000 gses leave heart for 10 minutes.
4, take water mutually, join 1/the 3 mols/L of 1000% physical volumes NaAc(pH 5.2), the icy cold ether of 2.500% physical volumes, mix evenly, -20 ℃ be 30 minutes.
5, 4 ℃ next 12000 gses leave heart for 15 minutes and carefully leave to ascend pure liquid, precipitate with 70% ether rinses, 4 ℃ next 12000 gses leave heart for 5 minutes.
6, be careful to leave to ascend pure liquid, precipitate the vacuum dry 5 minutes or air for dry 10 minutes, and dissolve in have no RNA Mao of double germ water or TE buffer liquid in.
7, take a 10 mls to carry on giving or get an electric shock Yong analysis, the another 10 mls useds for cDNA to synthesize.
[Notice]1, the whole operation should carry on under the low temperature possibly.
2, the virus RNA insets an inside at the coat protein, so want to well peel off virus coat protein and the general demand for many times carries on take out of Fen/chloroform to lift.
Section 4 cDNA synthesizes a technique
A, the Riboclone M-MLV(H- ) The cDNA synthesizes a technique
The RibocloneR M-MLV of Promega company(H- ) The cDNA synthesizes the system adoption M-MLV reversal to record the RNase H imperfection mutation stub of Mao to replace AMV reversal to record Mao and make synthetic cDNA longer.That system the first chain synthesize the usage M-MLV reversal to record Mao, cDNA the second chain synthesize adoption displacement to synthesize a method, adopt RNaseH and DNA to come together Mao Ⅰ to carry on displacement to synthesize, finally use T4 DNAs to come together Mao to slice to the single chain bitter end and the method is simple to easily go.That system tries to include:
20 particularities of μ g lead a thing
200 μ l M-MLVs the first chain buffer liquid(5 ×s), formula as follows: 250 mmols/L Tris ·Cl pH 8.3;(37 ℃ ) 375mmol/l KCl; 15mmol/L MgCl2; 50mmol/L DTT; The dATP, dCTP, dGTP of the 10 mmols/L, dTTP mixture(each 2.5 mmols/L)
2 ×s 625 the μ rRNasinR RNA Mao depressant
10,000 reversals of μ M-MLV record Mao, RNase H-
5 μ gs check against RNA
400 μ l M-MLVs the second chain buffer liquid(10 ×s), formula as follows:
400 mmols/L Tris ·Cl, pH 7.2; 850mmol/L KCl; 44mmol/L MgCl2;
30mmol/L DTT; 0.5 mgs/ml BSA.
500 μ RNase H
500 μ DNAs come together Mao Ⅰ
100 the μ T4 the DNAs come together Mao Ⅰ
2 ×s 1.25 mls not water with the nucleic acid Mao
Above all try in addition to check against RNA to need at-70 ℃ keep, the rest all can be keep in-20 ℃ , can synthesize 40 μ g mRNA.
(A) the first chain synthesize
1. Try
[α -32 Ps] dCTP(>400 Cis/mmol), EDTA(50 mMs and 200 mMs), TE-saturated Fen:chloroform(1:1), the Acs, ether(100% and 70%)s of the 7.5 M NH4, TE buffer liquid.
2. Operate a step
(1) take on putting out have no of germ RNA the eppendorf of the Mao a tube, join RNA template and adequacy to lead a thing, each time the μ g RNA use 0.5 μ gs to lead a thing(such as usage the Not Ⅰ lead a thing to deal with contact and use 0.3 μ gs), use H2 O adjustment a physical volume to 15 μ ls, 70 ℃ handle for 5 minutes, cool off to the indoor temperature, leave heart to make aqua the concentration is take care of bottom, again one by one in order join
5 ×s the first chain buffer liquid 5 μ ls
The Mao depressant of the rRNasin RNA 25 Us
M-MLV(H- )The reversal records Mao 200 Us
H2 Os adjust to total accumulate 25 μ ls
(2) by hand point to flip tube wall, absorb 5 μ ls to another an eppendorf a tube, join 2-5 μ Cis[α -32 Ps] dCTP(>400 Cis/mmol), in order to and the first chain isotope add a radio activity a measurement.
(3)37 ℃ (random lead a thing) or 42 ℃ (the other lead a thing) be Wen Yu4’s an hours
(4) take out to place at the ice
(5) add the eppendorf tube of measurement to join 95 the μ l 50 mM the EDTA terminate reaction, and make total accumulate to 100 μ ls.Commendable 90 μ lses carry on giving or get an electric shock Yong analysis(first take out to lift with the benzene Fen) and another 10 μ ls carry on isotope to add a radio activity a measurement.
The first chain synthesizes an eppendorf tube and can directly used for the second chain to synthesize
Note:Above 25 μ lses respond total accumulate medium the RNA use measure to one μ g, if synthesize 5 μ g RNA, then can pro rata extension respond a physical volume, pour 5 μ gs the RNA use 125 μ ls total accumulate to carry on synthesize.
(Two) the second chain synthesize
1, take the first chain to respond liquid 20 μ ls, one by one in order join again
10 ×s the second chain buffer liquid 20 μ ls
The DNA comes together Mao Ⅰ 23 μs
RNase H 0.8 μs
H2 Os add to eventually the physical volume is 100 μ ls
2, lightly mix evenly, if need to be carry on the second chain isotope add a radio activity measurement, commendable 10 μ ls go to another an eppendorf tube, join 2-5 μ Cis[α -32 Ps] dCTP.
3, 14 ℃s bathe for 2 hours.(if need cDNA of synthesize the longer than 3 kbs, then need to be prolong to 3-4 hours)
4, add to join in the measurement eppendorf tube 90 the μ l 50 mM EDTA, take 10 μ ls to carry on isotope to add a radio measurement, rest of can carry on giving or get an electric shock Yong analysis.
5, cDNA the second chain synthesize to leave a heart tube to respond a liquid 70 ℃ handles for 10 minutes, the low speed leaves heart to postpose ice up.
6, join 2 the μ T4 the DNAs come together Mao and 37 ℃s bathe for 10 minutes.
7, join 10 the μ l 200 mmols/L the EDTA terminate reaction.
8, use etc. physical volume Fen:the chloroform take out to lift a cDNA reaction liquid and leave heart for 2 minutes.
9, water mutually moves to another one eppendorf tube, join the 7.5 M acetic acid An(or the 1.5 M of 0.100% physical volumes sodium acetate, pH 5.2) of 0.500% physical volumes, after mixing evenly again join the icy cold ether(-20 ℃ ) of 2.500% physical volumes, -20 ℃ leaves heart for 5 minutes after place 30 minutes.
10, be careful to throw down up the pure liquid, join a 0.5 ml icy cold 70% ether, leave heart for 2 minutes.
11, be careful to move to ascend pure liquid, the aridity precipitate.
12, precipitate to dissolve in 10-20 the μ l TE buffer liquid.
(Three)the isotope add radio activity measurement, calculation and electricity Yong analysis
1. Try
The 1 mg/ml salmon Jing DNA, three chlorine acetic acid(TCA, 5% and 7%), alkaline Qiong fat sugar gum, alkaline gum electricity Yong buffer liquid,(30 mM NaOH, 1 mM EDTA) , 2 × sample buffer liquids, .(20 mM NaOH, 20% glycerin, 0.025% bromine Fens are blue)
2. Operate a step
(1) each take a 2(5) moderate two(4) reaction liquids each 3 μ ls, order at the glass fiber filter paper, the indoor temperature is dry, these samples represent total radio activity.
(2) the same be each to take to respond a liquid in 3 μ ls to imply 100 the μ l(1 mg/ml) salmon Jing DNA in, mix evenly, join 0.5 mls 5% TCAs, the whirlpool mixture instrument mixture postposes ice last 5-30 minuteses.
(3) filter with the glass fiber filter paper, wash with 5% icy cold TCAs three times, use 5 ml TCA each time, again wash with the 5 ml acetone or ether Piao, these sample representatives add radio activity.
(4) measurese total radio live strength respectively with add the radio live strength, can use cover Ge calculator, can also count with the liquid Shan.
3. The first chain yield measurese
The first chain adds a rate(%)= add cpm/total 100% of cpm ×
Add dNTP(nmol)=the 2 nmol dNTP/μ l × respond physical volume(μ l) ×( the first chain add a rate)
Establish 330 is the average molecular weight of each mol dNTP
Synthesize amount of cDNA(ng)=add the dNTP(nmol) × 330 ngs/nmol
MRNA to cDNA change rate=synthesize amount of cDNA(ng)/ the template RNA quantity(ng) × 100%
For example the total radio live strength is a 254,000 cpms, add the radio live strength as 3040 cpms, the RNA use touch the plank measure to one μ g, reaction physical volume is 25 μ ls, then:
Add a rate=3040/254000 the ×s be 100%s=1.2
Add amount of dNTP=the 2 nmol dNTP/μ l × 25 the ×s be 1.2%s=0.6 nmols
Synthesize amount of cDNA=the 330 ngs/nmol of the 0.6 nmol dNTP ×=198 ngs
MRNA to cDNA change rate=198 nms/1000 ng the × be 100%=19.8%
Because 1000 ng 20%(5 μ l/25 μ ls) in the RNA used for adding a measurement, but respond that the physical volume share total accumulate 80%, as a result physically and the first chain cDNA synthesize to measure to 0.8 ×s 198 ngs=158 ngs.
4. The second chain yield compute
In addition to need to be clean the first chain add dNTP, method together and the first chain yield calculation
The second chain adds a rate= add a radio activity/ total radio activity??
Add amount of dNTP(nnol)=[the 0.4 nmol dNTP/μ l × respond physical volume(μ l) － the first chain add nmol]× the second chain add a rate
The second chain cDNA synthesizes quantity(ng)=the × 330 ngs/nmol of the nmol dNTP
Double the chain cDNA change rate=double the chain cDNA synthesize quantity(ng)/ the single chain cDNA synthesize quantity(ng)
Example: The second chain adds the radio live strength as 2780 cmps, total radio live strength is 235000 cpms.
The second chain adds a rate=2780/235000 the ×s be 100%s=1.18%
The second chain synthesizes amount of dNTP=[(the 0.4 nmols/μ l × 100 μ ls)the － 0.48 nms]the × be 1.18%=0.47 nmols
Synthesize the second chain cDNA quantity(ng)=the × 330 ngs/nmol of the 0.47 nmol=155 ngs
Double the chain cDNA change rate=155 ngs/158 ng the × be 100%=98%
General cDNA the first chain change rate and double chain change rate take 12-50% and 50-200% as good.
(Four) the electricity Yong be analytical
The cDNA usually synthesized the first chain with the second chain the length needs to carry on 1.4% an alkaline Qiong fat sugar an electricity Yong for 350-6000 alkali Jis.Will the first chain with the second chain add measurement reaction liquid within tube to first take out to lift with the Fen, the ether precipitate, the method see this chapter(two) the second chain synthesize medium of 9-12, the first chain generally and with the second chain top kind quantity homology.
1. The standard molecular weight DNA accordings to the isotope marking of thing
(1) usually use the λ DNA/Hind Ⅲ part, come together Mao to carry on a 32 P marking with the Klenow DNA
10 the × Hind Ⅲ buffer liquid 2.5 μ ls
dATP 0.2mmol/L
dGTP 0.2mmol/L
[α -32 Ps]dCTP(400 Cis/mmol)2 μ Cis
The Ⅲ standard DNA of the λ DNA/Hind one μ g
The Klenow DNA comes together Mao one μ
Add H2 Os to arrive total accumulate 25 μ ls
(2) the indoor temperature place for 10 minutes and add 2.5 the μ l 200 mM the EDTA terminate reaction, join 2 × sample buffer liquids and store to save in-20 ℃ .
2. Give or get an electric shock Yong analysis
(1) use 50 mM NaCl, the 1 mM EDTA makes 1.4% alkaline Qiong fat sugar electricity Yong, apposition alkalescence electricity the Yong buffer be 30 minutes in liquid.
(2) sampling article liquid, use a TE adjustment physical volume, make the first chain with the second chain produce a rate measurement the physical volume homology of the liquid and join etc.2 × sample buffer liquids of the physical volume.(20 mmols/L NaOH, 20% glycerin, 0.025% bromine powder orchid)
(3) add kind, gave or get an electric shock Yong to the dyestuff to follow line before be apart from remain gum length of 1/3, the electricity Yong buffer liquid is 30 mmols/L NaOH, 1 mmol/L EDTA.
(4) dip the gum into 7% TCAs, the indoor temperature places for 30 minutes(until the dyestuff is change by the blue to the yellow) and take out to place a filter paper up, dry few hour.
(5) use the clingfilm package dry gum, the indoor temperature presses a X light slice.(use to increase feeling to hold-70 ℃ press a slice)
Two, other cDNAs synthesize a technique
The Riboclone M-MLV(H-) cDNA synthesizes a technique, besides which, ， the Promega company still provides several AMVs to synthesize to try a box, the dissimilarity tries what a box provide to lead a thing or leads a thing on postponing to deal with contact(Primpr-Adaptor) a dissimilarity, have oligo(dT)15 lead a thing and random lead a thing, Xba Ⅰ to lead a thing-postpone to deal with contact, the Not Ⅰ lead a thing-postpone to deal with contact etc., the rest tries one similar, these systems include:
The 20 mgs leads a thing
20 mls 5 ×s the first chain buffer liquid, formula as follows:
250 mmols/L Tris ·Cl, pH 8.3(42 ℃ );250 mmols/L KCl;50 mmols/L
MgCl2; The 2.5 mmols/L second Jing An(Spermidine); 50mmol/L DTT;
The dATP, dCTP, dGTP of the 20 mmols/L, dTTP(each 5 mmols/L).
The burnt carbonic acid sodium of the 50 mls 40 mMs
The 625 m Mao depressant of the rRNasin RNA
The 600 m AMV reversal records Mao
The 5 mgs 1.2 kbs checks against RNA
200 mls 10 ×s the second chain buffer liquid, formula as follows:
400 mmols/L Tris ·Cl, pH 7.2; 900mmol/L KCl; 30mmol/L
MgCl2; 30mmol/L DTT; 0.5 mgs/ml BSA.
2m E.Coli RNaseH
500m E.ColiDNA Polymerase Ⅰ
The 100 m T4 DNA Polymerase Ⅰ
1.5 mls not water with the nucleic acid Mao
Above all try in addition to check against RNA to need at-70 ℃ keep, the rest all can be keep in-20 ℃ , can synthesize 40 mgmRNAs.
(A) the first chain synthesize
What underneath introduction is a 25 ml physical volume respond system, that system can synthesize much to the 2 mgmRNAs, increase 1 mg mRNA each time, need to increase the reaction physical volume 10 mls, such as 5 mg the mRNA need a 55 mls to respond a physical volume.
Lead a thing or lead a thing-postpone to deal with contact to record Mao and mRNA with reversal of the comparison should keep for the 0.5 mgs/mg(lead a thing to the Not Ⅰ -postpone to deal with contact for the 0.3 mgs/mg) with 15 ms/mg respectively.
1, try:
[Ps a-32]dCTP(>400 cis/mmol), EDTA(50 mMs and 200 mMs), the TE saturated Mao/chloroform, the 7.5 mM NH4 Ac, 100% with 70% ether, TE buffer liquid.(10 mM Tris ·Cl, the pH 8.0,1 mm EDTA)
2, operate a step:
(1)take a put out have no of germ RNA the eppendorf tube of the Mao join of RNA sample, and lead a thing or lead a thing-postpone to deal with contact, use H2 Os to regulate a 0.5 mgs to lead the thing/mg mRNA(or the 0.3 mg Not Ⅰ lead a thing-postpone to deal with contact/mg mRNA) physical volume to the 15 mls, 70 ℃ heats for 5 minutes and treat cold to the indoor temperature leave heart for few second make the aqua concentration be take care of bottom, then one by one in order join:
5 ×s the first chain buffer liquid 5 mls
The Mao depressant of the rRNasinR RNA 25 mls
The 40 mMs burnt carbonic acid sodium 2.5 mls
The AMV reversal records the Mao 15 ms/mg RNA
Add to have no water RNA Mao water to total accumulate 25 mls
BE join burnt carbonic acid sodium and AMV reversal to record before the Mao, need reaction liquid 42 ℃s bathe for 5 minutes to keep burnt carbonic acid sodium from precipitate.The burnt carbonic acid sodium is mainly used for suppress the first chain formation hairpin structure.
(2)flip an eppendorf tube, take out a 5 mls to another an add at the 2-5 ms[Ps a-32]dCTP c>400 cis/mmol)(its physical volume can’t over the 1 ml), in order to and the first chain isotope add a radio activity a measurement.
(3)42 ℃s bathe for an hour.
(4)take out to place ice up.
(5)add the eppendorf tube of measurement to join 50 mM EDTA, terminate reaction, and make total accumulate to 100 mls.The commendable 90 mls carries on giving or get an electric shock Yong analysis and the another 10 mls carries on isotope to add a measurement.
(6) the first chain synthesize to leave a heart tube and can directly used for the second chain to synthesize.
(Two) the second chain synthesize
The second chain synthesize can in the first chain synthesize the reaction liquid directly carry on.
1, the first chain respond one by one in order join in the liquid(20 mls)
10 Xs the second chain buffer liquid 10 mls
E.The polymerase Ⅰ of the Coli DNA 23 ms
E.Coli RNaseH 0.8m
Fill with non-nuclear and sour Mao water to the total physical volume 100 mls
Rest step together this section 4 one 2-12(two) and(three) with.(four)
Thinking:
1. MRNA why withdraw is the key that the cDNA synthesizes success or failure?
2. Try comparison from lead leading method and displacement to synthesize the merit and shortcoming that the method synthesizes cDNA
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