CN110331191A - Using the method for Real-Time Fluorescent Quantitative PCR Technique detection Qian Beimayang CTSB gene organization express spectra - Google Patents
Using the method for Real-Time Fluorescent Quantitative PCR Technique detection Qian Beimayang CTSB gene organization express spectra Download PDFInfo
- Publication number
- CN110331191A CN110331191A CN201910794203.5A CN201910794203A CN110331191A CN 110331191 A CN110331191 A CN 110331191A CN 201910794203 A CN201910794203 A CN 201910794203A CN 110331191 A CN110331191 A CN 110331191A
- Authority
- CN
- China
- Prior art keywords
- ctsb
- real
- gene
- cell
- quantitative pcr
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of methods using Real-Time Fluorescent Quantitative PCR Technique detection Qian Beimayang CTSB gene organization express spectra.The present invention have the characteristics that it is easy to operate, rapidly and efficiently, sensibility height, high specificity, solve the problems, such as PCR pollution, PCR thermally labile, high degree of automation, than using other method applicabilities are stronger, effect is more preferable.And the present invention is easily understood, convenient for operation.
Description
Technical field
The present invention relates to bioscience technology field, it is related specifically to detect Qian Bei using Real-Time Fluorescent Quantitative PCR Technique
The method of Ma Yang CTSB gene organization express spectra.
Background technique
CTSB (Cathepsin B, cathepsin B) belongs to lysosomal cysteine inhibitor protein enzyme family member,
It is closely connected from different biochemical function and Pathological barrier.Such as under the conditions of appropriate pH < 7, degradation is played in lysosome
Albumen promotes the biological functions such as Apoptosis.Under extracellular pH suitable condition, its biological function, such as macrophage can be also played
Cell, osteoclast, fibroblast and other noble cells.Some researches show that cathepsins in people and Mice Body,
CTSB is necessary to embryo's normal development and uterine decidua；Also studies have reported that CTSB, CTSD etc. are in endometrium or gestation
It is expressed in the conceptus of early stage sheep, there are also others research shows that CTSB gene is also expressed in the ovarian follicle of mouse, it is seen then that
The development of CTSB gene pairs gonadal tissue plays a role.
Real-time fluorescence quantitative PCR is one kind in DNA amplification reaction, surveys each polymerase chain reaction with fluorescent chemical
The method of product total amount after should recycling.The specific dna sequence in sample to be tested is quantitatively divided by internal reference or outer ginseng method
The method of analysis.It is, by fluorescence signal, to be measured in real time to PCR process during PCR amplification.Due in PCR amplification
There are linear relationships for the starting copy number of exponential time base, the Ct value of template and the template, so becoming quantitative foundation.With tradition
Round pcr compared to Real-Time Fluorescent Quantitative PCR Technique efficiently solve traditional quantitative can only end point determination limitation, realize
Each round circulation detects the intensity of first order fluorescence signal, and is recorded among computer software, by each sample Ct value
It calculates, quantitative result is obtained according to standard curve.Therefore, real-time fluorescence quantitative PCR without internal standard be built upon two bases it
On.The research of forefathers is it has been shown that CTSB gene pairs sheep follicular development has a certain impact, but its mechanism is not known；
Using Real-Time Fluorescent Quantitative PCR Technique, express spectra of the numb sheep CTSB gene in Guizhou Province north in gonadal tissue is established, for explaining CTSB
Expressional function mechanism of the gene in the numb sheep different tissues in Guizhou Province north has certain directive function.
Summary of the invention
For the deficiency of existing invention material, Guizhou Province is detected using Real-Time Fluorescent Quantitative PCR Technique the present invention provides a kind of
The method of Bei Mayang CTSB gene organization express spectra, it have it is easy to operate, rapidly and efficiently, it is sensibility height, high specificity, effective
Solve the problems, such as the features such as PCR pollution, PCR thermally labile, high degree of automation, more stronger than using other method applicabilities,
Effect is more preferable.
In order to achieve the above object, the present invention is achieved by the following technical programs: using Real-Time Fluorescent Quantitative PCR Technique
The method for detecting Qian Beimayang CTSB gene organization express spectra, includes the following steps:
(1) the numb sheep ewe hypothalamus in Guizhou Province north, the hypophysis, uterus, defeated ovum for extracting the single lamb of oestrus production after butchering, producing more lambs
The total serum IgE of pipe and 5 kinds of gonadal tissues of ovary；It is total using ultramicrospectrophotometer and 1% agarose gel electrophoresis Detection and Extraction
The specificity of RNA；
(2) the first chain of cDNA is synthesized according to Reverse Transcriptase kit to the satisfactory total serum IgE of extraction；And with reverse transcription
The first chain of cDNA afterwards is template, carries out regular-PCR amplification to target gene CTSB and reference gene β-actin respectively；
(3) sequence of target gene CTSB upstream primer is 5'-TGACTCGCATGTA GGTTGC-3', downstream primer
Sequence is 5'-TGGAGACGCTGTAGGAA-3'；The sequence of reference gene β-actin upstream primer is 5'-
AGATGTGGATCAGCAAGC AG-3', downstream primer sequence be 5'-CCAATCTCATCTCGTTTTCTG-3'；With 1.5%
Agarose gel electrophoresis is detected.
(4) purpose band that will test out is cut, and gel extraction product is connect with pMD19-T carrier, is then converted to large intestine
Bacillus DH5 α competent cell will be accredited as positive bacterium solution and be sequenced, correct bacterium solution will be sequenced and carry out after the screening of blue hickie
Expand numerous and plasmid to extract, and the recombinant plasmid of acquisition is subjected to quantitative dilution, using the recombinant plasmid after diluting as template, uses
Real-time fluorescence quantitative PCR amplifying target genes CTSB and reference gene β-actin obtains standard curve and melting curve；It utilizes
2- Δ Δ Ct relative quantification method calculates the relative expression quantity of target gene CTSB, and detects differential expression amount.
When being expanded in step (2) to target gene CTSB and reference gene β-actin, PCR program are as follows: initial denaturation
95 DEG C of 5min, 95 DEG C of denaturation 30s, 55 DEG C of annealing 45s, 72 DEG C of extension 30s, 72 DEG C extend 7min eventually；Reaction system is 20 μ L,
In include: 10 μ L of 2x Taq PCR Master Mix, 2 μ L of DNA profiling, upstream and downstream primer each 1 μ L, ddH2O 6μL。
The system that gel extraction product and pMD-19T carrier are attached in step (4) is 10 μ L, including
pMDTM1 μ L of 19-T Vector, 4 μ L, Solution I ligase of gel extraction product, 5 μ L, 16 DEG C of metal baths connect 12h.
The method that competent escherichia coli cell is converted into step (4) is as follows:
1) bacillus coli DH 5 alpha competent cell is placed in after thawing on ice to jiggle and cell suspends；
2) the connection liquid that metal bath stays overnight connection is added in bacillus coli DH 5 alpha competent cell, and gently will with pipette tips
Cell is mixed with liquid is connect, and placement stands 20min on ice；
3) heated at constant temperature 90s in 42 DEG C of water-baths is being put it into, cooled on ice 10min is placed after heating；
4) common liq culture medium is added into test tube, placement is shaken with 37 DEG C in case, and 200rpm vibrates 1h；
5) body fluid after vibrating is put into 5000rpm in refrigerated centrifuge and is centrifuged 5min, and centrifugation bottom of the tube has cell precipitation, uses
Pipette tips sop up supernatant, blow and beat remaining culture medium back and forth with pipette tips and mix cell and culture medium；
6) cell suspension is uniformly coated in the solid medium tablets containing AMP containing X-gal and IPTG；
7) plate is placed into 1h in 37 DEG C of insulating boxs, culture 12-16h is inverted after planar surface liquid is dry.
The present invention compared with the existing technology, the present invention have it is easy to operate, rapidly and efficiently, sensibility height, high specificity, have
Effect solves the problems, such as the features such as PCR pollution, PCR thermally labile, high degree of automation, than using other method applicabilities more
By force, effect is more preferable.And the present invention is easily understood, convenient for operation.
Specific embodiment
Embodiment: using the method for Real-Time Fluorescent Quantitative PCR Technique detection Qian Beimayang CTSB gene organization express spectra；
TRIzol Reagent kit is purchased from Guizhou Lv Meng Ying Chuan Biotechnology Co., Ltd；Liquid nitrogen, chloroform, 0.5%TAE buffering
Liquid, isopropanol, 75% ethyl alcohol agarose, are commercially available；Fluorescent quantitation reagent UltraSYBR Mixture is purchased from and holds up biology section, section
Skill Co., Ltd；Plastic recovery kit, miniplasmids extracts kit, LB culture medium, Reverse Transcriptase kit are purchased from Beijing Kang Weishi
Discipline Biotechnology Co., Ltd.Ultraviolet-uisible spectrophotometer is purchased from U.S. Thermo Fisher Co., Ltd；Electrophoresis apparatus
(DYY-2C type) is purchased from Beijing Liuyi Instrument Factory；PCR amplification instrument (C1000 TouchTM), real-time fluorescence quantitative PCR instrument (type
Number be CFX96 Real-Time System), gel imaging system (Universal Hood II), be purchased from U.S. BIO-RAD
Co., Ltd.
One, the extraction of Guizhou Province north numb sheep hypothalamus, hypophysis, uterus, fallopian tubal, ovary tissue total serum IgE:
(1) centrifuge tube of 1.5mL is marked according to different groups, 1mL Trizol is added in every pipe.
(2) it takes suitable tissue sample in the good mortar of washing and sterilizing for every group, liquid nitrogen is added in tissue abrasion, as far as possible
Sufficiently, the powder of about 60mg is added in grinding in each centrifuge tube.
(3) centrifuge tube is acutely shaken and mixes 1-2min, juxtaposition stands 15min at room temperature sufficiently to crack tissue sample
Product.
(4) 200 μ L chloroform solns are added in every pipe, 20s are mixed by inversion rapidly, and stand 15-20min, after standing
4 DEG C are put into high speed freezing centrifuge, and 12000rpm is centrifuged 15min.
(5) supernatant is taken after being centrifuged, about 500 μ L is taken to be put into a new centrifuge tube.
(6) the frost isopropanol of 500 μ L is added in every pipe, is mixed by inversion 15s, stands 10min on ice, with 13000rpm, 4
DEG C, it is centrifuged 10min, abandons supernatant, precipitating is total serum IgE.
(7) every pipe is added 75% ethanol solution of 1mL frost, washing precipitating RNA, with supercentrifuge with 7500rpm,
4 DEG C of centrifugation 5min abandon supernatant.
(8) ethanol solution extra in pipe abandon is inhaled, superclean bench is placed in and dries, about 30min.
(9) the RNAse-free water of 30 μ L is added in every pipe, is stored at room temperature 5min, dissolution RNA precipitating.
(10) the dissolved RNA sample of 1 μ L is taken, with the concentration and purity of the total serum IgE of ultramicrospectrophotometer Detection and Extraction,
2 μ LRNA samples are separately taken to carry out agarose gel electrophoresis detection, remaining total serum IgE is saved backup in -80 DEG C of refrigerators.
Two, the synthesis of the first chain of cDNA
Using Transcriptor First Strand cDNA Synthesis Kit Reverse Transcriptase kit by extraction
RNA reverse transcription synthesizes the first chain of cDNA.Reverse transcription system be 20 μ L, including 4 μ L of dNTP mix 2.5mmEach,
2 μ L of Primer mix, 1 μ L of RNA template, 5 × RT Buffer, 4 μ L, 2 μ L of DTT 0.1m, 1 μ L of HiFi Script, supplement
RNase-Free water 6μL.PCR reaction condition is 42 DEG C of incubation 50min, and 85 DEG C of incubation 5min are saved in -20 DEG C of refrigerators.
PCR product is detected using 1.5% agarose gel electrophoresis.
Three, the design and synthesis of primer
According to goat Capra hircus (goat) CTSB gene order (accession number: XM_ issued on GenBank
018051779.1)；Primer information is shown in Table 1.Choose the reference gene that β-actin is this test.Primer transfers to the raw work in Shanghai raw
The synthesis of object engineering services limited liability company.
1 primer details of table
Four, the foundation of PCR amplification and melting curve
(1) when being expanded to target gene CTSB and reference gene β-actin, PCR program are as follows: 95 DEG C of initial denaturation
5min, 95 DEG C of denaturation 30s, 55 DEG C of annealing 45s, 72 DEG C of extension 30s, 72 DEG C extend 7min eventually；Reaction system is 20 μ L, wherein
It include: 10 μ L of 2x Taq PCR Master Mix, 2 μ L of DNA profiling, upstream and downstream primer each 1 μ L, ddH2O 6μL。
(2) it by the cutting of the purpose band of PCR product and weighing in test tube, and is added according to the two volumes of glue weight
Buffer PG solution, 50 DEG C of heating water bath 2-3min and mixing of turning upside down are presented until blob of viscose is in vitro completely melt
Acquired solution is transferred to 13000rpm in adsorption column after yellow liquid and is centrifuged 1min, the liquid in collecting pipe is discarded, will adsorb
Buffer PW, the 13000rpm room temperature centrifugation 1min of 450 μ L is added in column, outwells the liquid in collecting pipe, repeats the above steps
Once, with 1300rpm room temperature be centrifuged 1min, outwell the liquid in collecting pipe, by adsorption column be put into a new 1.5mL from
In heart pipe, the Buffer EB solution of 30-50 μ L is added into adsorption column, 13000rpm room temperature is centrifuged 1min, and the solution of collection is
For cDNA solution.- 20 DEG C are placed in save or for follow-up test
(3) DNA solution obtained after gel extraction and pMD-19T carrier being attached, system is 10 μ L, including
pMDTM1 μ L of 19-T Vector, 4 μ L, Solution I ligase of gel extraction product, 5 μ L, 16 DEG C of metal baths connect 1-12h.
(4) by connect liquid be converted into competent escherichia coli cell method it is as follows: 1. by 100 μ L bacillus coli DH 5 alphas
Competent cell is placed in thaw on ice after jiggle cell suspend.2. large intestine is added in the connection liquid that metal bath stays overnight connection
In bacillus DH5 α competent cell, and gently cell is mixed with liquid is connect with pipette tips, placement stands 20min on ice.3. will
It is put into heated at constant temperature 90s in 42 DEG C of water-baths, and cooled on ice 10min is placed after heating.4. it is common that 600 μ L are added into test tube
Fluid nutrient medium, placement are shaken with 37 DEG C in case, and 200rpm vibrates 1h.5. the body fluid after oscillation is put into refrigerated centrifuge
5000rpm is centrifuged 5min, it is seen that centrifugation bottom of the tube has cell precipitation, sops up 500 μ L supernatants with pipette tips, is blown and beaten back and forth with pipette tips
Remaining culture medium mixes cell and culture medium.Contain AMP containing X-Gal and IPTG 6. being uniformly coated in cell suspension
Solid medium tablets on.7. plate is placed 1h in 37 DEG C of insulating boxs, culture 12- is inverted after planar surface liquid is dry
16h。
(5) after plate is inverted culture 12-16h, white colony is picked out in 5mL fluid nutrient medium with ampicillin
In, and be put into shaking table and shake 37 DEG C of culture 12-16h of bacterium, to liquid muddiness, after regular-PCR and agarose gel electrophoresis detection,
Positive bacterium solution containing target fragment is sent to Shanghai Sheng Gong bioengineering Co., Ltd to be sequenced and is identified, will identify correct bacterium solution
It carries out expanding the extraction of numerous and plasmid, by the recombinant plasmid dilution after extraction in order to real-time fluorescence quantitative PCR amplification.
Five, the optimization of real time fluorescence quantifying PCR method
Real-Time Fluorescent Quantitative PCR Technique detect CTSB gene specific as a result, it has been found that.It is contaminated using SYBR Green I fluorescence
Material method carries out real-time fluorescence quantitative PCR reaction and 3 repetitions is arranged, and the amplification curve of generation is smooth, S-type, overall trend one
It causes, clip size is consistent, and amplification property is preferable.Melting curve is in unimodal, and peak value is preferable, nothing but specific amplification products and primer two
Aggressiveness, without mixed and disorderly, distortion band.Prove that CTSB gene specific is good, result of study has convincingness.
Six, detection CTSB gene is in the numb sheep hypothalamus of single lamb and more lamb Guizhou Provinces north, hypophysis, uterus, 5 fallopian tubal, ovary groups
Expression quantity in knitting
Real-time fluorescence quantitative PCR reaction is carried out using SYBR Green I fluorescent dye determination.Real-time fluorescence quantitative PCR reaction
System is 10 μ L, including 1 μ L of cDNA template, SYBR Premix Ex Taq TM enzyme (2 ×) 5.5 μ L, upstream and downstream primer
Each 0.5 μ L, ddH2O 2.5 μL.Quantitative fluorescent PCR response procedures are as follows: 95 DEG C of initial denaturation 1min, 95 DEG C of denaturation 15 s, and 55 DEG C
Anneal 15s, and 60 DEG C of extension 32s, 39 circulations, 3 repetitions are arranged in each sample, and negative control is arranged in each test,
Detect the practical expression that CTSB gene is organized in hypothalamus, hypophysis, uterus, fallopian tubal and 5, ovary.Utilize 2- Δ Δ Ct
The relative expression quantity of relative quantification method calculating target gene: 2- Δ Δ Ct=2- [(purpose sample Ct- reference gene Ct)-(control
Sample Ct- reference gene Ct)], single lamb, more lamb samples are using the numb sheep inferior colliculus cerebral tissue in Guizhou Province north as standard.Use SPSS 20.0
One-Way ANOVA carries out one-way analysis of variance in software, carries out significance test of difference using LSD method, test result is adopted
It is indicated with mean+SD, it is extremely significant using P < 0.01 as difference with P < 0.05 for significance of difference judgment criteria
The judgment criteria of property.
The present invention is not limited to above-mentioned preferred forms, and other forms can be obtained in anyone under the inspiration of the present invention
Product.But no matter make any variation in terms of catalyst composition, structure and proportion, it is all have it is identical with the application or
Similar technical solution, all belongs to the scope of protection of the present invention.
Sequence table
<110>Guizhou University
<120>using the method for Real-Time Fluorescent Quantitative PCR Technique detection Qian Beimayang CTSB gene organization express spectra
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>goat (Nelumbo nucifera)
<400> 1
tgactcgcat gtaggttgc 19
<210> 2
<211> 17
<212> DNA
<213>goat (Nelumbo nucifera)
<400> 2
tggagacgct gtaggaa 17
<210> 3
<211> 20
<212> DNA
<213>goat (Nelumbo nucifera)
<400> 3
agatgtggat cagcaagcag 20
<210> 4
<211> 21
<212> DNA
<213>goat (Nelumbo nucifera)
<400> 4
ccaatctcat ctcgttttct g 21
Claims (4)
1. a kind of method using Real-Time Fluorescent Quantitative PCR Technique detection Qian Beimayang CTSB gene organization express spectra, feature
It is: includes the following steps:
(1) extract the oestrus after butchering produce single lamb, the numb sheep ewe hypothalamus in Guizhou Province north for producing more lambs, hypophysis, uterus, fallopian tubal and
The total serum IgE of 10 gonadal tissues of ovary；Using ultramicrospectrophotometer and 1% agarose gel electrophoresis Detection and Extraction total serum IgE
Specificity, integrality and purity；
(2) the first chain of cDNA is synthesized according to Reverse Transcriptase kit to the satisfactory total serum IgE of extraction；And with reverse transcription after
The first chain of cDNA is template, carries out regular-PCR amplification to target gene CTSB and reference gene β-actin respectively；
(3) sequence of target gene CTSB upstream primer is 5'-TGACTCGCATGTAGGTTGC-3', the sequence of downstream primer is
5'-TGGAGACGCTGTAGGAA-3'；The sequence of reference gene β-actin upstream primer is 5'-
AGATGTGGATCAGCAAGCAG-3', downstream primer sequence be 5'-CCAATCTCATCTCGTTTTCTG-3'；PCR amplification produces
Object is detected using 1.5% agarose gel electrophoresis.
(4) purpose band that electrophoresis detection goes out is cut, gel extraction product is connect with pMD19-T carrier, is then converted to large intestine
The bacterium solution for being accredited as positive is sequenced after the screening of blue hickie, correct bacterium solution will be sequenced by bacillus DH5 α competent cell
Expand it is numerous extract work with plasmid, and the recombinant plasmid of acquisition is subjected to quantitative dilution, using the recombinant plasmid after diluting as
Template is obtained standard curve and is melted using real-time fluorescence quantitative PCR amplifying target genes CTSB and reference gene β-actin
Curve；The relative expression quantity of target gene CTSB is calculated using 2- Δ Δ Ct relative quantification method, and detects differential expression amount.
2. according to claim 1 using the expression of Real-Time Fluorescent Quantitative PCR Technique detection Qian Beimayang CTSB gene organization
The method of spectrum, it is characterised in that: when being expanded in step (2) to target gene CTSB and reference gene β-actin, PCR journey
Sequence are as follows: 95 DEG C of 5min of initial denaturation, 95 DEG C of denaturation 30s, 55 DEG C of annealing 45s, 72 DEG C of extension 30s, 72 DEG C extend 7min eventually；Reactant
System is 20 μ L, including: 2x Taq PCR Master Mix 10 μ L, 2 μ L of DNA profiling, each 1 μ L of upstream and downstream primer,
ddH2O 6μL。
3. according to claim 1 using the expression of Real-Time Fluorescent Quantitative PCR Technique detection Qian Beimayang CTSB gene organization
The method of spectrum, it is characterised in that: the system that gel extraction product and pMD-19T carrier are attached in step (4) is 10 μ L,
In include pMDTM1 μ L of 19-T Vector, 4 μ L, Solution I ligase of gel extraction product, 5 μ L, 16 DEG C of metal bath connections
12h。
4. according to claim 1 using the expression of Real-Time Fluorescent Quantitative PCR Technique detection Qian Beimayang CTSB gene organization
The method of spectrum, it is characterised in that: the method that competent escherichia coli cell is converted into step (4) is as follows:
1) bacillus coli DH 5 alpha competent cell is placed in after thawing on ice to jiggle and cell suspends；
2) the connection liquid that metal bath stays overnight connection is added in bacillus coli DH 5 alpha competent cell, and with pipette tips gently by cell
It is mixed with liquid is connect, placement stands 20min on ice；
3) heated at constant temperature 90s in 42 DEG C of water-baths is being put it into, cooled on ice 10min is placed after heating；
4) common liq culture medium is added into test tube, placement is shaken with 37 DEG C in case, and 200rpm vibrates 1h；
5) body fluid after vibrating is put into 5000rpm in refrigerated centrifuge and is centrifuged 5min, and centrifugation bottom of the tube has cell precipitation, uses pipette tips
Supernatant is sopped up, remaining culture medium is blown and beaten back and forth with pipette tips and mixes cell and culture medium；
6) cell suspension is uniformly coated in the solid medium tablets containing AMP containing X-gal and IPTG；
7) plate is placed into 1h in 37 DEG C of insulating boxs, culture 12-16h is inverted after planar surface liquid is dry.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910794203.5A CN110331191A (en) | 2019-08-27 | 2019-08-27 | Using the method for Real-Time Fluorescent Quantitative PCR Technique detection Qian Beimayang CTSB gene organization express spectra |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910794203.5A CN110331191A (en) | 2019-08-27 | 2019-08-27 | Using the method for Real-Time Fluorescent Quantitative PCR Technique detection Qian Beimayang CTSB gene organization express spectra |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110331191A true CN110331191A (en) | 2019-10-15 |
Family
ID=68150172
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910794203.5A Pending CN110331191A (en) | 2019-08-27 | 2019-08-27 | Using the method for Real-Time Fluorescent Quantitative PCR Technique detection Qian Beimayang CTSB gene organization express spectra |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110331191A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114015709A (en) * | 2021-11-04 | 2022-02-08 | 塔里木大学 | Application of sheep CTSD gene in regulation and control of initiation of primitive phase |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1373223A (en) * | 2001-09-30 | 2002-10-09 | 山东大学 | Cathepsin B gene of bollworm and its clone, recombination and expression techniques |
| WO2004105573A2 (en) * | 2003-05-21 | 2004-12-09 | The Wistar Institute Of Anatomy And Biology | Method of diagnosis of cancer based on gene expression profiles in cells |
| CA2671270A1 (en) * | 2006-12-29 | 2008-07-17 | Asuragen, Inc. | Mir-16 regulated genes and pathways as targets for therapeutic intervention |
| CN104805214A (en) * | 2015-05-14 | 2015-07-29 | 贵州大学 | Establishment method for Guizhou north Pockmarked goat FSHR (Follicle Simulating Hormone Receptor) gene tissue expression spectrums based on real-time fluorescence quantitative PCR (Polymerase Chain Reaction) |
-
2019
- 2019-08-27 CN CN201910794203.5A patent/CN110331191A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1373223A (en) * | 2001-09-30 | 2002-10-09 | 山东大学 | Cathepsin B gene of bollworm and its clone, recombination and expression techniques |
| WO2004105573A2 (en) * | 2003-05-21 | 2004-12-09 | The Wistar Institute Of Anatomy And Biology | Method of diagnosis of cancer based on gene expression profiles in cells |
| CA2671270A1 (en) * | 2006-12-29 | 2008-07-17 | Asuragen, Inc. | Mir-16 regulated genes and pathways as targets for therapeutic intervention |
| CN104805214A (en) * | 2015-05-14 | 2015-07-29 | 贵州大学 | Establishment method for Guizhou north Pockmarked goat FSHR (Follicle Simulating Hormone Receptor) gene tissue expression spectrums based on real-time fluorescence quantitative PCR (Polymerase Chain Reaction) |
Non-Patent Citations (5)
| Title |
|---|
| MENG-HELIU等: "Excessive expression and activity of cathepsin B in sheep cumulus cells compromises oocyte developmental competence", 《SMALL RUMINANT RESEARCH》 * |
| XU FENG等: "Genome-wide differential expression profiling of mRNAs and lncRNAs associated with prolificacy in Hu sheep", 《BIOSCI REP》 * |
| 周志楠等: "黔北麻羊CTSB、CTSD基因的克隆、生物信息学分析及在不同组织中的表达", 《中国兽医学报》 * |
| 祁昕: "绵羊CTSB基因克隆及其活性与***发育能力相关性研究", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
| 祁昕等: "组织蛋白酶对羊***发育能力影响的研究进展", 《中国草食动物科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114015709A (en) * | 2021-11-04 | 2022-02-08 | 塔里木大学 | Application of sheep CTSD gene in regulation and control of initiation of primitive phase |
| CN114015709B (en) * | 2021-11-04 | 2023-04-25 | 塔里木大学 | Application of sheep CTSD gene in regulation and control of primordial initiation |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104531884B (en) | Primer and probe composition for distinguishing various animal sources in colla corii asini, kit and multiple real-time fluorescence quantification PCR detection method | |
| Li et al. | Culture and characterization of chicken small intestinal crypts | |
| CN109295186A (en) | A kind of method based on genome sequencing detection adenine single base editing system undershooting-effect and its application in gene editing | |
| Jia et al. | Genetic variation of mitochondrial D-loop region and evolution analysis in some Chinese cattle breeds | |
| Lin et al. | A multiplex PCR assay for species identification of raw and cooked bonito | |
| CN106868224A (en) | The RT PCR methods of detection pig blue-ear disease poison classical strainses, highly pathogenic mutant strain and the strains of NADC 30 | |
| CN109136389A (en) | A kind of DNA bar code standard detection segment CO II, kit and its application method identifying American cockroach | |
| CN109439775A (en) | A kind of multi-PCR detection method of porcine pathogen | |
| CN110331191A (en) | Using the method for Real-Time Fluorescent Quantitative PCR Technique detection Qian Beimayang CTSB gene organization express spectra | |
| CN101481742B (en) | Detection kit for Mycoplasma hyopneumoniae and use thereof | |
| CN100371459C (en) | Kit with fluorescent quantitative RT-PCR detection technique used for cell keratin 19(Ck19)mRNA | |
| JP7090357B2 (en) | Use of primer sets, kits, and microRNA serum markers and primer sets to identify the sex of sturgeon | |
| CN103320516A (en) | Method and special product for assisted identification of swine backfat thickness character | |
| CN108085417A (en) | A kind of yak coronavirus detection kit and application based on constant temperature isolation type fluorescent PCR platform | |
| CN1712547A (en) | Fluorescent quantitative RT-PCR detecting kit of 2-f(o)etoprotein (AFP)mRNA | |
| CN101463352B (en) | DNA fragment related to pig intramuscular fat deposition and use thereof | |
| Batra et al. | Molecular cloning and characterization of Mx2 for early pregnancy diagnosis in Bubalus bubalis | |
| CN111041084A (en) | Detection kit for small fat Willi syndrome and use method thereof | |
| CN109055587A (en) | Detect kit, specific primer and probe and the application of E. CoIi content | |
| CN106399521B (en) | One-step method identifies primer combination and its identification method, kit of sika deer and products thereof the multiplex PCR of gender | |
| CN106868217A (en) | A kind of detection primer of zika virus and application | |
| CN112852980A (en) | Detection method for detecting nucleic acid of Atlantic salmon based on CRISPR fluorescence method and detection kit thereof | |
| CN105950717A (en) | Quantitative determination method for donkey and bovine derived ingredients in colla corii asini liquid semi-finished product or finished product, composition and kit | |
| CN105219878A (en) | A kind of neosporosis detection kit | |
| CN110317900A (en) | A kind of the multiple RT-PCR primer sets and detection method of quick detection CSFV, GETV, JEV |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20191015 |
|
| RJ01 | Rejection of invention patent application after publication |