CN102876677A - Double-stranded RNA (ribonucleic acid) preparation method - Google Patents
Double-stranded RNA (ribonucleic acid) preparation method Download PDFInfo
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- CN102876677A CN102876677A CN2012103682910A CN201210368291A CN102876677A CN 102876677 A CN102876677 A CN 102876677A CN 2012103682910 A CN2012103682910 A CN 2012103682910A CN 201210368291 A CN201210368291 A CN 201210368291A CN 102876677 A CN102876677 A CN 102876677A
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Abstract
The invention discloses a double-stranded RNA (ribonucleic acid) preparation method. An existing in-vitro siRNA preparation method is high in price, long in custom period and limited in experiment size. The double-stranded RNA preparation method includes: cloning a target gene molecule by a PCR (polymerase chain reaction) amplification method; using pET-T7 plasmids to construct an expression vector including target gene segments; transforming the pET-T7 expression vector including gene segment insertion to an expressive strain E.coliHT115, and selecting a positive bacterial colony including a target vector; inoculating to an LB (Luria-Bertani) fluid medium and adding IPTG (isopropyl-beta-d-thiogalactoside) for induction and culture; extracting total RNA, dissolving to RNaseA buffer fluid for denaturation, cooling for renaturation, adding RNaseA for digestion, removing single-stranded RNA, purifying double-stranded RNA, and dissolving precipitation, so that the obtained solution, namely the purified double-stranded RNA is obtained. The double-stranded RNA preparation method is simple, rapid in implementation steps, high in operating repeatability and unlimited in sample synthesis quantity.
Description
Technical field
The present invention relates to a kind of preparation method of double-stranded RNA, specifically utilize Escherichia coli fermentation to prepare the double-stranded RNA sample, the RNA that this two strands sample can be used as crustacean disturbs.
Background technology
RNA disturbs (RNAi) to refer to the sequence-specific gene silencing phenomenon of being brought out by double-stranded RNA on a kind of molecular biology, and its mechanism is by the translation that hinders specific gene or transcribes inhibition of gene expression.When the double-stranded RNA that imports in the cell with endogenous mRNA coding region homology, degraded occurs and causes genetic expression reticent in this mRNA, is the effective means of the multiple biological gene function of research.The RNAi technology is prevented method as emerging gene, is widely applied in fields such as functional genomics, microbiology, the researchs of gene expression regulation mechanism.
At present, mainly utilize siRNA (siRNA) to carry out gene and disturb, in the biologies such as Arabidopis thaliana, beautiful new rhabditis axei, drosophila melanogaster, zebra fish and mouse, use.Present stage, the method for the external siRNA of preparation respectively has relative merits: chemical synthesis process can prepare high-quality siRNA, but price is high, and the customization cycle is long; The in-vitro transcription method is synthesized siRNA, and cost is relatively low, but scale and the amount of experiment are restricted.
The immunity system of crustacean is the open cycle system, does not have immunoglobulin (Ig), lacks antibody-mediated immune response, and its immunologic mechanism is take non-specific immunity as main.Therefore, the long-chain double-stranded RNA of external synthetic can enter in the animal body, reticent goal gene, and downward modulation corresponding protein expression level is carried out the phenotype research of certain gene function disappearance fast and economically.
Utilize Escherichia coli fermentation to prepare the double-stranded RNA sample, a large amount of long-chain double-stranded RNAs of the production of efficient quick had both shortened the required preparation cycle of chemosynthesis at short notice, had reduced the expense of great number, had broken through again the scale restriction of in-vitro transcription.
Summary of the invention
The objective of the invention is for the deficiencies in the prior art, a kind of preparation method of double-stranded RNA is provided, the method is suitable for using when crustacean RNA disturbs.
The present invention is that to solve the problems of the technologies described above the technical scheme steps of taking as follows:
Step (1). gene clone: the method clone goal gene molecule that utilizes pcr amplification.
According to the cDNA sequences Design of goal gene molecule forward primer and the reverse primer with restriction enzyme site.Take the cDNA sequence of goal gene molecule as template, carry out pcr amplification, clone's goal gene molecule obtains the PCR product.The PCR product is analyzed with agarose gel electrophoresis.Adopt PCR product purification test kit to carry out purifying, obtain the PCR product behind the purifying.
Step (2). vector construction: utilize the pET-T7 plasmid construction to contain the expression vector of goal gene fragment.
PCR product and pET-T7 plasmid behind the purifying carry out double digestion with restriction enzyme respectively, and the enzyme that obtains goal gene is cut the enzyme of product and pET-T7 plasmid and cut product.The enzyme that the enzyme of goal gene is cut product and pET-T7 plasmid is cut product and is reclaimed the test kit purifying with gel, obtains the enzyme of goal gene behind the purifying and cuts behind product and the purifying enzyme of pET-T7 plasmid and cut product.Utilizing dna ligation kit to connect the enzyme of goal gene behind the purifying cuts behind product and the purifying enzyme of pET-T7 plasmid and cuts product.Change the product after connecting over to competent cell
E. coliAmong the DH5 α, coated plate, 30～37
oC cultivated 12～24 hours.Select positive single bacterium colony and carry out PCR detection, agarose gel electrophoresis analysis.Choose the positive single bacterium colony that contains the goal gene fragment, sequencing analysis inserts the exactness of gene fragment order, obtains containing single bacterium colony that the correct gene fragment is inserted.
Step (3). carrier transforms:
Picking contains single bacterium colony that the correct gene fragment is inserted through evaluation, is inoculated in the LB liquid nutrient medium 30～37
oCultivated centrifugal being precipitated under the C condition 12～24 hours.Utilize the pET-T7 expression vector in a small amount of plasmid extraction test kit extracting precipitation thalline, the detected through gel electrophoresis extract product.The pET-T7 expression vector that will contain the gene fragment insertion is transformed into the expression type bacterial strain
E.coliAmong the HT115, coated plate is cultivated, and selects positive bacteria and drops into the performing PCR detection, and gel electrophoresis analysis is chosen the positive bacterium colony that contains the purpose carrier, preserves stand-by.The positive bacterium colony that contains the purpose carrier herein is to carry the pET-T7 carrier
E.coliThe single bacterium colony of HT115.
Step (4). the abduction delivering of double-stranded RNA:
The pET-T7 carrier will be carried
E.coliThe single colony inoculation of HT115 in the LB liquid nutrient medium, 30～37
oCultivated under the C condition 12～24 hours.With the ratio enlarged culturing of 1:20～1:50 to OD
600=0.4～1.0.Add isopropyl-β-D-thiogalactoside(IPTG) IPTG solution in the LB liquid nutrient medium, adding to the final concentration of IPTG in the LB liquid nutrient medium is 0.2～1.0mmol/L, 30～37
oC inducing culture 2～8 hours.Centrifugal collection bacterial precipitation, the phosphoric acid buffer rinsing obtains the bacterial precipitation after the rinsing, preserves stand-by.
Described LB liquid nutrient medium is the aqueous solution that contains 10g/l Tryptones, 5g/l yeast extract, 10g/l sodium-chlor and 50 μ g/ml penbritins, and pH is 7.4;
Described yeast extract adopts conventional currently available products, can obtain through the market purchase;
Described phosphoric acid buffer is for containing 8g/l sodium chloride nacl, 0.2g/l potassium chloride (KCl), 1.38g/l Sodium phosphate dibasic Na
2HPO
4, 0.24g/l potassium primary phosphate KH
2PO
4The aqueous solution, pH is 7.4.
Step (5). the purifying of double-stranded RNA:
Total RNA extracting: get the bacterial precipitation after the rinsing, utilize the total RNA in the bacterial precipitation after the Trizol test kit extracts rinsing;
Sex change: total RNA that will extract is dissolved in ribonuclease A (RNase A) damping fluid, and 80～90
oC sex change 5～15 minutes obtains denaturing soln;
Renaturation: place water-bath to be cooled to normal temperature denaturing soln, so that the denaturing soln renaturation, obtain the solution after the renaturation;
Enzyme is cut: add RNase A in the solution after renaturation and carry out enzyme and cut, the final concentration that adds in the solution of RNase A after renaturation is 10～50 μ g/ml, 35～37
oReacted under the C condition 10～60 minutes, and removed single stranded RNA, obtain enzyme and cut after product, the electrophoresis detection enzyme is cut effect;
Purifying: cut in the after product to add with enzyme at enzyme and cut the phenol chloroformic solution of product equivalent volumes, abundant mixing, centrifuging and taking supernatant liquor.The dehydrated alcohol that in supernatant liquor, adds 2～3 times of supernatant liquor volumes, fully mixing leaves standstill, precipitation double-stranded RNA, centrifugal collecting precipitate; Fully wash centrifugal collecting precipitation, seasoning with 70～75 ﹪ ethanol;
Dissolving: add the aqua sterilisa dissolution precipitation, gained solution is the double-stranded RNA sample behind the purifying.Detected through gel electrophoresis double-stranded RNA sample, light splitting range meter method is measured nucleic acid samples concentration ,-30～-20
oC preserves stand-by.
Described ribonuclease A damping fluid is the aqueous solution of the Tutofusin tris hydrochloric acid Tris-HCl of the sodium-acetate NaAc, 10 mmol/L that contain 300 mmol/L, and pH is 8.0.
Described phenol chloroformic solution is the by volume mixed solution that mixes of 0.8:1～1.2:1 of the acid saturated phenol of pH 4.0～6.0 and chloroform.
The beneficial effect that the present invention has:
The present invention utilizes Escherichia coli fermentation to prepare the double-stranded RNA sample, and a large amount of double-stranded RNA samples of the production of efficient quick can shorten preparation cycle at short notice, reduce again high cost, and the restriction of the scale when having broken through in-vitro transcription.This invention scope of application is extensive, can synthesize the double-stranded RNA of different length fragments.Simultaneously, this invention preparation method is simple, and implementation step is quick, and operation repeatability is strong, and the sample resultant quantity is unrestricted.
Embodiment
The below is further analyzed the present invention.
Green fluorescent protein GFP is a kind of biological marker commonly used.During the RNA interference experiment, the interference of GFP gene is often used as control experiment group data.To take green fluorescence protein gene as example, elaborate the synthetic and preparation method of double-stranded RNA in the present embodiment.
Embodiment 1
Step (1). gene clone: the method clone GFP gene molecule that utilizes pcr amplification.
According to the cDNA sequences Design of the GFP gene of having reported in gene pool forward primer GFP-F and the reverse primer GFP-R with restriction enzyme site.Take the cDNA of GFP gene molecule as template, carry out pcr amplification, clone's goal gene molecule obtains the PCR product.
The PCR reaction system is:
10×PCR buffer 2.5μl
25mM Mg
2+ 1.5μl
5mM dNTPs 1μl
10μM GFP-PF 1μl
10μM GFP-PR 1μl
GFP cDNA 1μl
Taq archaeal dna polymerase 0.25 μ l
Adding water to final volume is 25 μ l.
The pcr amplification reaction condition is: elder generation 94
oThen C sex change 5 minutes carries out 30 amplification cycles, last 72
oC extended 10 minutes; Wherein amplification cycles is 94
oC sex change 30 seconds, 50
oC annealing 30 seconds, 72
oC extended 1 minute.
The PCR product is analyzed with 1.0 ﹪ agarose gel electrophoresis.Adopt PCR product purification test kit to carry out purifying, obtain the PCR product of the GFP gene behind the purifying.
Step (2). vector construction: utilize the pET-T7 plasmid construction to contain the expression vector of GFP gene fragment.
In the present embodiment with restriction enzyme
XbaI and
EcoRI is example, elaborates pET-T7 Vector construction process.
PCR product and pET-T7 plasmid behind the purifying are all used restriction enzyme
XbaI and
EcoRI carries out double digestion, and the enzyme that obtains the GFP gene is cut the enzyme of product and pET-T7 plasmid and cut product.The double digestion reaction system is as follows:
PET-T7 plasmid double digestion reaction system:
PET-T7 plasmid 1 μ g
10×M buffer 1μl
XbaI 1μl
EcoRI 1μl
Add water to final volume 20 μ l, 37
oC reaction 8 hours, the enzyme that obtains the pET-T7 plasmid is cut product.
PCR product double digestion reaction system:
PCR product 1 μ g
10×M buffer 1μl
XbaI 1μl
EcoRI 1μl
Add water to final volume 20 μ l, 37
oC reaction 8 hours, the enzyme that obtains the GFP gene is cut product.
The enzyme that the enzyme of GFP gene is cut product and pET-T7 plasmid is cut product and is reclaimed the test kit purifying with gel, obtains the enzyme of GFP gene behind the purifying and cuts behind product and the purifying enzyme of pET-T7 plasmid and cut product.
Utilizing dna ligation kit to connect the enzyme of GFP gene behind the purifying cuts behind product and the purifying enzyme of pET-T7 plasmid and cuts product, 16
oConnect 4 hours under the C condition.Change the product after connecting over to competent cell
E. coliAmong the DH5 α, coated plate, 30
oC cultivated 24 hours.Select positive single bacterium colony from flat board and carry out the PCR detection, primer is GFP-PF and GFP-PR, and the PCR reaction template is the bacterium liquid that contains single bacterium colony, and the pcr amplification reaction condition is same as above, analyzes with 1.0 ﹪ agarose gel electrophoresis.Choose the positive single bacterium colony that contains the GFP gene fragment, sequencing analysis inserts the exactness of gene fragment order, obtains containing single bacterium colony that correct GFP gene fragment is inserted.
Step (3). carrier transforms:
Picking contains single bacterium colony of correct GFP gene insertion sequence through evaluation, is inoculated in the 1ml LB liquid nutrient medium 30
oC cultivated 24 hours, 5000 rev/mins, centrifugal 5 minutes, was precipitated.
Utilize the pET-T7 expression vector in a small amount of plasmid extraction test kit extracting precipitation thalline, 1 ﹪ agarose gel electrophoresis detects extract product.
The pET-T7 expression vector that will contain the insertion of GFP gene fragment is transformed into the expression type bacterial strain
E.coliAmong the HT115, coated plate is cultivated, and selects positive bacteria and drops into the performing PCR detection, and reaction conditions is same as above.The PCR product is analyzed with 1.0 ﹪ agarose gel electrophoresis.Choose the positive bacterium colony that contains the purpose carrier, preserve stand-by.
Herein the positive bacterium colony that contains the purpose carrier is the pET-T7 carrier that carries the GFP gene fragment
E.coliThe single bacterium colony of HT115.
Step (4). the abduction delivering of double-stranded RNA:
The pET-T7 carrier of GFP gene fragment will be carried
E.coliThe single colony inoculation of HT115 in the LB liquid nutrient medium, 30
oCultivated 24 hours under the C condition.The pET-T7 carrier of GFP gene fragment will be carried
E.coliThe single bacterium colony of HT115 is transferred to enlarged culturing in the fresh LB liquid nutrient medium, and the volume ratio of original LB liquid nutrient medium and fresh LB liquid nutrient medium is 1:20, cultivates to make bacterial growth to OD under similarity condition
600=0.4.Add isopropyl-β-D-thiogalactoside(IPTG) IPTG solution in the LB liquid nutrient medium, adding to the final concentration of IPTG in the LB liquid nutrient medium is 0.2mmol/L, 30
oC inducing culture 8 hours.5000 rev/mins, centrifugal 5 minutes, collect bacterial precipitation.Phosphoric acid buffer rinsing 2 times obtains the bacterial precipitation after the rinsing, preserves stand-by.
Step (5). the purifying of double-stranded RNA:
Total RNA extracting: get the bacterial precipitation after the rinsing, utilize the total RNA in the bacterial precipitation after the Trizol test kit extracts rinsing;
Sex change: total RNA that will extract is dissolved in ribonuclease A (RNase A) damping fluid, and 80
oC sex change 15 minutes obtains denaturing soln;
Renaturation: place water-bath to be cooled to normal temperature denaturing soln, so that the denaturing soln renaturation, obtain the solution after the renaturation;
Enzyme is cut: add RNase A in the solution after renaturation and carry out enzyme and cut, the final concentration that adds in the solution of RNase A after renaturation is 10 μ g/ml, 35
oSingle stranded RNA is removed in C reaction 60 minutes, obtains enzyme and cuts after product, detects enzyme with 1.0 ﹪ agarose gel electrophoresis and cuts effect;
Purifying: cut in the after product to add with enzyme at enzyme and cut the phenol chloroformic solution of after product equivalent volumes, abundant mixing, 10000 rev/mins, 4
oC, 20 minutes, the centrifuging and taking supernatant liquor.Add the dehydrated alcohol of 2 times of supernatant liquor volumes in supernatant liquor, fully mixing leaves standstill, precipitation double-stranded RNA, 10000 rev/mins, 4
oC, 30 minutes, centrifugal collecting precipitate; Fully wash with 70 ﹪ ethanol, 10000 rev/mins, 4
oC, 20 minutes, centrifugal collecting precipitation, seasoning;
Dissolving: add the aqua sterilisa dissolution precipitation, gained solution is the double-stranded RNA sample behind the purifying.Utilize 1.0 ﹪ agarose gel electrophoresis to detect the double-stranded RNA sample, light splitting range meter method is measured nucleic acid samples concentration ,-30
oC preserves stand-by.
Above-mentioned LB liquid nutrient medium is for containing the 10g/l Tryptones, the 5g/l yeast extract, and the aqueous solution of 10g/l sodium-chlor and 50 μ g/ml penbritins, pH is 7.4;
Phosphoric acid buffer is for containing the 8g/l sodium chloride nacl, 0.2g/l potassium chloride (KCl), 1.38g/l Sodium phosphate dibasic Na
2HPO
4, 0.24g/l potassium primary phosphate KH
2PO
4The aqueous solution, pH is 7.4;
The ribonuclease A damping fluid is the sodium-acetate NaAc that contains 300mmol/L, the aqueous solution of the Tutofusin tris hydrochloric acid Tris-HCl of 10mmol/L, and pH is 8.0;
The phenol chloroformic solution is the by volume mixed solution that mixes of 0.8:1 of the acid saturated phenol of pH 4.0 and chloroform.
Embodiment 2
Step (1). gene clone: the method clone GFP gene molecule that utilizes pcr amplification.
According to the cDNA sequences Design of the GFP gene of having reported in gene pool forward primer GFP-F and the reverse primer GFP-R with restriction enzyme site.Take the cDNA of GFP gene molecule as template, carry out pcr amplification, clone's goal gene molecule obtains the PCR product.
The PCR reaction system is:
10×PCR buffer 2.5μl
25mM Mg
2+ 1.5μl
5mM dNTPs 1μl
10μM GFP-PF 1μl
10μM GFP-PR 1μl
GFP cDNA 1μl
Taq archaeal dna polymerase 0.25 μ l
Adding water to final volume is 25 μ l.
The pcr amplification reaction condition is: elder generation 94
oThen C sex change 5 minutes carries out 38 amplification cycles, last 72
oC extended 10 minutes; Wherein amplification cycles is 94
oC sex change 30 seconds, 55
oC annealing 30 seconds, 72
oC extended 2 minutes.
The PCR product is analyzed with 1.5 ﹪ agarose gel electrophoresis.Adopt PCR product purification test kit to carry out purifying, obtain the PCR product of the GFP gene behind the purifying.
Step (2). vector construction: utilize the pET-T7 plasmid construction to contain the expression vector of GFP gene fragment.
In the present embodiment with restriction enzyme
XbaI and
EcoRI is example, elaborates pET-T7 Vector construction process.
PCR product and pET-T7 plasmid behind the purifying are all used restriction enzyme
XbaI and
EcoRI carries out double digestion, and the enzyme that obtains the GFP gene is cut the enzyme of product and pET-T7 plasmid and cut product.The double digestion reaction system is as follows:
PET-T7 plasmid double digestion reaction system:
PET-T7 plasmid 1 μ g
10×M buffer 1μl
XbaI 1μl
EcoRI 1μl
Add water to final volume 20 μ l, 37
oC reaction 16 hours, the enzyme that obtains the pET-T7 plasmid is cut product.
PCR product double digestion reaction system:
PCR product 1 μ g
10×M buffer 1μl
XbaI 1μl
EcoRI 1μl
Add water to final volume 20 μ l, 37
oC reaction 16 hours, the enzyme that obtains the GFP gene is cut product.
The enzyme that the enzyme of GFP gene is cut product and pET-T7 plasmid is cut product and is reclaimed the test kit purifying with gel, obtains the enzyme of GFP gene behind the purifying and cuts behind product and the purifying enzyme of pET-T7 plasmid and cut product.
Utilizing dna ligation kit to connect the enzyme of GFP gene behind the purifying cuts behind product and the purifying enzyme of pET-T7 plasmid and cuts product, 16
oConnect 12 hours under the C condition.Change the product after connecting over to competent cell
E. coliAmong the DH5 α, coated plate, 37
oC cultivated 12 hours.Select positive single bacterium colony from flat board and carry out the PCR detection, primer is GFP-PF and GFP-PR, and the PCR reaction template is the bacterium liquid that contains single bacterium colony, and reaction conditions is same as above, analyzes with 1.5 ﹪ agarose gel electrophoresis.Choose the positive single bacterium colony that contains the GFP gene fragment, sequencing analysis inserts the exactness of gene fragment order, obtains containing single bacterium colony that correct GFP gene fragment is inserted.
Step (3). carrier transforms.
Picking contains single bacterium colony of correct GFP gene insertion sequence through evaluation, is inoculated in the 5ml LB liquid nutrient medium 37
oC cultivated 12 hours, 5000 rev/mins, centrifugal 10 minutes, was precipitated.
Utilize the pET-T7 expression vector in a small amount of plasmid extraction test kit extracting precipitation thalline, 1 ﹪ agarose gel electrophoresis detects extract product.
The pET-T7 expression vector that will contain the insertion of GFP gene fragment is transformed into the expression type bacterial strain
E.coliAmong the HT115, coated plate is cultivated, and selects positive bacteria and drops into the performing PCR detection, and reaction conditions is same as above.The PCR product is analyzed with 1.5 ﹪ agarose gel electrophoresis.Choose the positive bacterium colony that contains the purpose carrier, preserve stand-by.
Herein the positive bacterium colony that contains the purpose carrier is the pET-T7 carrier that carries the GFP gene fragment
E.coliThe single bacterium colony of HT115.
Step (4). the abduction delivering of double-stranded RNA:
The pET-T7 carrier of GFP gene fragment will be carried
E.coliThe single colony inoculation of HT115 in the LB liquid nutrient medium, 37
oCultivated 12 hours under the C condition.The pET-T7 carrier of GFP gene fragment will be carried
E.coliThe single bacterium colony of HT115 is transferred to enlarged culturing in the fresh LB liquid nutrient medium, and the volume ratio of original LB liquid nutrient medium and fresh LB liquid nutrient medium is 1:50, cultivates to make bacterial growth to OD under similarity condition
600=1.0.Add isopropyl-β-D-thiogalactoside(IPTG) IPTG solution in the LB liquid nutrient medium, adding to the final concentration of IPTG in the LB liquid nutrient medium is 1.0mmol/L, 37
oC inducing culture 2 hours.5000 rev/mins, 10 minutes centrifugal, collects bacterial precipitation.Phosphoric acid buffer rinsing 2 times obtains the bacterial precipitation after the rinsing, preserves stand-by.
Step (5). the purifying of double-stranded RNA:
Total RNA extracting: get the bacterial precipitation after the rinsing, utilize the total RNA in the bacterial precipitation after the Trizol test kit extracts rinsing;
Sex change: total RNA that will extract is dissolved in ribonuclease A (RNase A) damping fluid, and 90
oC sex change 5 minutes obtains denaturing soln;
Renaturation: place water-bath to be cooled to normal temperature denaturing soln, so that the denaturing soln renaturation, obtain the solution after the renaturation;
Enzyme is cut: add RNase A in the solution after renaturation and carry out enzyme and cut, the final concentration that adds in the solution of RNase A after renaturation is 50 μ g/ml, 37
oSingle stranded RNA is removed in C reaction 10 minutes, obtains enzyme and cuts after product, detects enzyme with 1.5 ﹪ agarose gel electrophoresis and cuts effect;
Purifying: cut in the after product to add with enzyme at enzyme and cut the phenol chloroformic solution of after product equivalent volumes, abundant mixing, 11000 rev/mins, 4
oC, 13 minutes, the centrifuging and taking supernatant liquor.Add the dehydrated alcohol of 3 times of supernatant liquor volumes in supernatant liquor, fully mixing leaves standstill, precipitation double-stranded RNA, 11000 rev/mins, 4
oC, 25 minutes, centrifugal collecting precipitate; Fully wash with 75 ﹪ ethanol, 11000 rev/mins, 4
oC, 13 minutes, centrifugal collecting precipitation, seasoning;
Dissolving: add the aqua sterilisa dissolution precipitation, gained solution is the double-stranded RNA sample behind the purifying.Utilize 1.2 ﹪ agarose gel electrophoresis to detect the double-stranded RNA sample, light splitting range meter method is measured nucleic acid samples concentration ,-20
oC preserves stand-by.
Above-mentioned LB liquid nutrient medium is for containing the 10g/l Tryptones, the 5g/l yeast extract, and the aqueous solution of 10g/l sodium-chlor and 50 μ g/ml penbritins, pH is 7.4;
Phosphoric acid buffer is for containing the 8g/l sodium chloride nacl, 0.2g/l potassium chloride (KCl), 1.38g/l Sodium phosphate dibasic Na
2HPO
4, 0.24g/l potassium primary phosphate KH
2PO
4The aqueous solution, pH is 7.4;
The ribonuclease A damping fluid is the sodium-acetate NaAc that contains 300mmol/L, the aqueous solution of the Tutofusin tris hydrochloric acid Tris-HCl of 10mmol/L, and pH is 8.0;
The phenol chloroformic solution is the by volume mixed solution that mixes of 1.2:1 of the acid saturated phenol of pH 6.0 and chloroform.
Embodiment 3
Step (1). gene clone: the method clone GFP gene molecule that utilizes pcr amplification.
According to the cDNA sequences Design of the GFP gene of having reported in gene pool forward primer GFP-F and the reverse primer GFP-R with restriction enzyme site.Take the cDNA of GFP gene molecule as template, carry out pcr amplification, clone's goal gene molecule obtains the PCR product.
The PCR reaction system is:
10×PCR buffer 2.5μl
25mM Mg
2+ 1.5μl
5mM dNTPs 1μl
10μM GFP-PF 1μl
10μM GFP-PR 1μl
GFP cDNA 1μl
Taq archaeal dna polymerase 0.25 μ l
Adding water to final volume is 25 μ l.
The pcr amplification reaction condition is: elder generation 94
oThen C sex change 5 minutes carries out 35 amplification cycles, last 72
oC extended 10 minutes; Wherein amplification cycles is 94
oC sex change 30 seconds, 52
oC annealing 30 seconds, 72
oC extended 1.5 minutes.
The PCR product is analyzed with 1.2 ﹪ agarose gel electrophoresis.Adopt PCR product purification test kit to carry out purifying, obtain the PCR product of the GFP gene behind the purifying.
Step (2). vector construction: utilize the pET-T7 plasmid construction to contain the expression vector of GFP gene fragment.
In the present embodiment with restriction enzyme
XbaI and
EcoRI is example, elaborates pET-T7 Vector construction process.
PCR product and pET-T7 plasmid behind the purifying are all used restriction enzyme
XbaI and
EcoRI carries out double digestion, and the enzyme that obtains the GFP gene is cut the enzyme of product and pET-T7 plasmid and cut product.The double digestion reaction system is as follows:
PET-T7 plasmid double digestion reaction system:
PET-T7 plasmid 1 μ g
10×M buffer 1μl
XbaI 1μl
EcoRI 1μl
Add water to final volume 20 μ l, 37
oC reaction 12 hours, the enzyme that obtains the pET-T7 plasmid is cut product.
PCR product double digestion reaction system:
PCR product 1 μ g
10×M buffer 1μl
XbaI 1μl
EcoRI 1μl
Add water to final volume 20 μ l, 37
oC reaction 12 hours, the enzyme that obtains the GFP gene is cut product.
The enzyme that the enzyme of GFP gene is cut product and pET-T7 plasmid is cut product and is reclaimed the test kit purifying with gel, obtains the enzyme of GFP gene behind the purifying and cuts behind product and the purifying enzyme of pET-T7 plasmid and cut product.
Utilizing dna ligation kit to connect the enzyme of GFP gene behind the purifying cuts behind product and the purifying enzyme of pET-T7 plasmid and cuts product, 16
oConnect 8 hours under the C condition.Change the product after connecting over to competent cell
E. coliAmong the DH5 α, coated plate, 35
oC cultivated 18 hours.Select positive single bacterium colony from flat board and carry out the PCR detection, primer is GFP-PF and GFP-PR, and the PCR reaction template is the bacterium liquid that contains single bacterium colony, and reaction conditions is same as above, analyzes with 1.2 ﹪ agarose gel electrophoresis.Choose the positive single bacterium colony that contains the GFP gene fragment, sequencing analysis inserts the exactness of gene fragment order, obtains containing single bacterium colony that correct GFP gene fragment is inserted.
Step (3). carrier transforms.
Picking contains single bacterium colony of correct GFP gene insertion sequence through evaluation, is inoculated in the 3ml LB liquid nutrient medium 35
oC cultivated 18 hours, 5000 rev/mins, centrifugal 8 minutes, was precipitated.
Utilize the pET-T7 expression vector in a small amount of plasmid extraction test kit extracting precipitation thalline, 1 ﹪ agargel electrophoresis detects extract product.
The pET-T7 expression vector that will contain the insertion of GFP gene fragment is transformed into the expression type bacterial strain
E.coliAmong the HT115, coated plate is cultivated, and selects positive bacteria and drops into the performing PCR detection, and reaction conditions is same as above.The PCR product is analyzed with 1.2 ﹪ agarose gel electrophoresis.Choose the positive bacterium colony that contains the purpose carrier, preserve stand-by.
Herein the positive bacterium colony that contains the purpose carrier is the pET-T7 carrier that carries the GFP gene fragment
E.coliThe single bacterium colony of HT115.
Step (4). the abduction delivering of double-stranded RNA:
The pET-T7 carrier of GFP gene fragment will be carried
E.coliThe single colony inoculation of HT115 in the LB liquid nutrient medium, 35
oCultivated 15 hours under the C condition.The pET-T7 carrier of GFP gene fragment will be carried
E.coliThe single bacterium colony of HT115 is transferred to enlarged culturing in the fresh LB liquid nutrient medium, and the volume ratio of original LB liquid nutrient medium and fresh LB liquid nutrient medium is 1:30, cultivates to make bacterial growth to OD under similarity condition
600=0.7.Add isopropyl-β-D-thiogalactoside(IPTG) IPTG solution in the LB liquid nutrient medium, adding to the final concentration of IPTG in the LB liquid nutrient medium is 0.6mmol/L, 35
oC inducing culture 5 hours.5000 rev/mins, 8 minutes centrifugal, collects bacterial precipitation.Phosphoric acid buffer rinsing 2 times obtains the bacterial precipitation after the rinsing, preserves stand-by.
Step (5). the purifying of double-stranded RNA:
Total RNA extracting: get the bacterial precipitation after the rinsing, utilize the total RNA in the bacterial precipitation after the Trizol test kit extracts rinsing;
Sex change: total RNA that will extract is dissolved in ribonuclease A (RNase A) damping fluid, and 85
oC sex change 10 minutes obtains denaturing soln;
Renaturation: place water-bath to be cooled to normal temperature denaturing soln, so that the denaturing soln renaturation, obtain the solution after the renaturation;
Enzyme is cut: add RNase A in the solution after renaturation and carry out enzyme and cut, the final concentration that adds in the solution of RNase A after renaturation is 30 μ g/ml, 36
oSingle stranded RNA is removed in C reaction 30 minutes, obtains enzyme and cuts after product, detects enzyme with 1.2 ﹪ agarose gel electrophoresis and cuts effect;
Purifying: cut in the after product to add with enzyme at enzyme and cut the phenol chloroformic solution of after product equivalent volumes, abundant mixing, 12000 rev/mins, 4
oC, 10 minutes, the centrifuging and taking supernatant liquor.Add the dehydrated alcohol of 2.5 times of supernatant liquor volumes in supernatant liquor, fully mixing leaves standstill, precipitation double-stranded RNA, 12000 rev/mins, 4
oC, 15 minutes, centrifugal collecting precipitate; Fully wash with 73 ﹪ ethanol, 12000 rev/mins, 4
oC, 10 minutes, centrifugal collecting precipitation, seasoning;
Dissolving: add the aqua sterilisa dissolution precipitation, gained solution is the double-stranded RNA sample behind the purifying.Utilize 1.2 ﹪ agarose gel electrophoresis to detect the double-stranded RNA sample, light splitting range meter method is measured nucleic acid samples concentration ,-25
oC preserves stand-by.
Above-mentioned LB liquid nutrient medium is for containing the 10g/l Tryptones, the 5g/l yeast extract, and the aqueous solution of 10g/l sodium-chlor and 50 μ g/ml penbritins, pH is 7.4;
Phosphoric acid buffer is for containing the 8g/l sodium chloride nacl, 0.2g/l potassium chloride (KCl), 1.38g/l Sodium phosphate dibasic Na
2HPO
4, 0.24g/l potassium primary phosphate KH
2PO
4The aqueous solution, pH is 7.4;
The ribonuclease A damping fluid is the sodium-acetate NaAc that contains 300mmol/L, the aqueous solution of the Tutofusin tris hydrochloric acid Tris-HCl of 10mmol/L, and pH is 8.0;
The phenol chloroformic solution is the by volume mixed solution that mixes of 1:1 of the acid saturated phenol of pH 5.0 and chloroform.
Claims (5)
1. the preparation method of a double-stranded RNA is characterized in that the method may further comprise the steps:
Step (1). gene clone: the method clone goal gene molecule that utilizes pcr amplification;
According to the cDNA sequences Design of goal gene molecule forward primer and the reverse primer with restriction enzyme site; Take the cDNA sequence of goal gene molecule as template, carry out pcr amplification, clone's goal gene molecule obtains the PCR product; The PCR product is analyzed with agarose gel electrophoresis; Adopt PCR product purification test kit to carry out purifying, obtain the PCR product behind the purifying;
Step (2). vector construction: utilize the pET-T7 plasmid construction to contain the expression vector of goal gene fragment;
PCR product and pET-T7 plasmid behind the purifying carry out double digestion with restriction enzyme respectively, and the enzyme that obtains goal gene is cut the enzyme of product and pET-T7 plasmid and cut product; The enzyme that the enzyme of goal gene is cut product and pET-T7 plasmid is cut product and is reclaimed the test kit purifying with gel, obtains the enzyme of goal gene behind the purifying and cuts behind product and the purifying enzyme of pET-T7 plasmid and cut product; Utilizing dna ligation kit to connect the enzyme of goal gene behind the purifying cuts behind product and the purifying enzyme of pET-T7 plasmid and cuts product; Change the product after connecting over to competent cell
E. coliAmong the DH5 α, coated plate, 30～37
oC cultivated 12～24 hours; Select positive single bacterium colony and carry out PCR detection, agarose gel electrophoresis analysis; Choose the positive single bacterium colony that contains the goal gene fragment, sequencing analysis inserts the exactness of gene fragment order, obtains containing single bacterium colony that the correct gene fragment is inserted;
Step (3). carrier transforms:
Picking contains single bacterium colony that the correct gene fragment is inserted through evaluation, is inoculated in the LB liquid nutrient medium 30～37
oCultivated centrifugal being precipitated under the C condition 12～24 hours; Utilize the pET-T7 expression vector in a small amount of plasmid extraction test kit extracting precipitation thalline, the detected through gel electrophoresis extract product; The pET-T7 expression vector that will contain the gene fragment insertion is transformed into the expression type bacterial strain
E.coliAmong the HT115, coated plate is cultivated, and selects positive bacteria and drops into the performing PCR detection, and gel electrophoresis analysis is chosen the positive bacterium colony that contains the purpose carrier, preserves stand-by; The positive bacterium colony that contains the purpose carrier herein is to carry the pET-T7 carrier
E.coliThe single bacterium colony of HT115;
Step (4). the abduction delivering of double-stranded RNA:
The pET-T7 carrier will be carried
E.coliThe single colony inoculation of HT115 in the LB liquid nutrient medium, 30～37
oCultivated under the C condition 12～24 hours; With the ratio enlarged culturing of 1:20～1:50 to OD
600=0.4～1.0; Add isopropyl-β-D-thiogalactoside(IPTG) IPTG solution in the LB liquid nutrient medium, adding to the final concentration of IPTG in the LB liquid nutrient medium is 0.2～1.0mmol/L, 30～37
oC inducing culture 2～8 hours; Centrifugal collection bacterial precipitation, the phosphoric acid buffer rinsing obtains the bacterial precipitation after the rinsing, preserves stand-by;
Step (5). the purifying of double-stranded RNA:
Total RNA extracting: get the bacterial precipitation after the rinsing, utilize the total RNA in the bacterial precipitation after the Trizol test kit extracts rinsing;
Sex change: total RNA that will extract is dissolved in the ribonuclease A damping fluid, and 80～90
oC sex change 5～15 minutes obtains denaturing soln;
Renaturation: place water-bath to be cooled to normal temperature denaturing soln, so that the denaturing soln renaturation, obtain the solution after the renaturation;
Enzyme is cut: add ribonuclease A in the solution after renaturation and carry out enzyme and cut, the final concentration that adds in the solution of ribonuclease A after renaturation is 10～50 μ g/ml, 35～37
oReacted under the C condition 10～60 minutes, and removed single stranded RNA, obtain enzyme and cut after product, the electrophoresis detection enzyme is cut effect;
Purifying: cut in the after product to add with enzyme at enzyme and cut the phenol chloroformic solution of product equivalent volumes, abundant mixing, centrifuging and taking supernatant liquor; The dehydrated alcohol that in supernatant liquor, adds 2～3 times of supernatant liquor volumes, fully mixing leaves standstill, precipitation double-stranded RNA, centrifugal collecting precipitate; Fully wash centrifugal collecting precipitation, seasoning with 70～75 ﹪ ethanol;
Dissolving: add the aqua sterilisa dissolution precipitation, gained solution is the double-stranded RNA sample behind the purifying; Detected through gel electrophoresis double-stranded RNA sample, light splitting range meter method is measured nucleic acid samples concentration ,-30～-20
oC preserves stand-by.
2. a kind of preparation method of double-stranded RNA as claimed in claim 1, it is characterized in that step (3) and the described LB liquid nutrient medium of step (4) are for containing the 10g/l Tryptones, the 5g/l yeast extract, the aqueous solution of 10g/l sodium-chlor and 50 μ g/ml penbritins, pH is 7.4.
3. a kind of preparation method of double-stranded RNA as claimed in claim 1 is characterized in that the described phosphoric acid buffer of step (4) is for containing the 8g/l sodium chloride nacl, 0.2g/l potassium chloride (KCl), 1.38g/l Sodium phosphate dibasic Na
2HPO
4, 0.24g/l potassium primary phosphate KH
2PO
4The aqueous solution, pH is 7.4.
4. a kind of preparation method of double-stranded RNA as claimed in claim 1, it is characterized in that the described ribonuclease A damping fluid of step (5) is the sodium-acetate NaAc that contains 300 mmol/L, the aqueous solution of the Tutofusin tris hydrochloric acid Tris-HCl of 10 mmol/L, pH is 8.0.
5. a kind of preparation method of double-stranded RNA as claimed in claim 1 is characterized in that the described phenol chloroformic solution of step (5) is the by volume mixed solution that mixes of 0.8:1～1.2:1 of the acid saturated phenol of pH 4.0～6.0 and chloroform.
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| CN104745628A (en) * | 2015-04-16 | 2015-07-01 | 浙江万里学院 | Method for sex induction of hermaphrodite shrimps |
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| CN114774448A (en) * | 2022-04-11 | 2022-07-22 | 浙江理工大学 | Preparation method of functional single-stranded self-assembled RNA (ribonucleic acid) nanoparticles |
| CN114891782A (en) * | 2022-05-24 | 2022-08-12 | 四川农业大学 | Method for purifying RNA by phenol chloroform method |
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| CN113151335A (en) * | 2021-05-27 | 2021-07-23 | 上海市农业科学院 | Rapid preparation method of microbial double-stranded RNA |
| CN114774448A (en) * | 2022-04-11 | 2022-07-22 | 浙江理工大学 | Preparation method of functional single-stranded self-assembled RNA (ribonucleic acid) nanoparticles |
| CN114891782A (en) * | 2022-05-24 | 2022-08-12 | 四川农业大学 | Method for purifying RNA by phenol chloroform method |
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