CN104988134A - Method for rapidly preparing DNA Ladder with low cost - Google Patents
Method for rapidly preparing DNA Ladder with low cost Download PDFInfo
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- CN104988134A CN104988134A CN201510265744.0A CN201510265744A CN104988134A CN 104988134 A CN104988134 A CN 104988134A CN 201510265744 A CN201510265744 A CN 201510265744A CN 104988134 A CN104988134 A CN 104988134A
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Abstract
The invention provides a method for rapidly preparing DNA Ladder with low cost. The method comprises the steps that strips needed by the DNA Ladder is obtained through a PCR amplification method, the size and concentration of all the strips can be precisely controlled and are not limited by enzyme digestion loci and plasmid concentration of a traditional method (such as EcoR I enzyme digestion lambda DNA), and the production cost can be reduced by 90%; and fragments containing the target size are constructed onto a pMD18-T carrier, a universal primer on the carrier is utilized for conducting PCR amplification on each fragment, all the DNA fragments of the DNA Ladder are diluted and mixed according to the designed concentration, and finally the DNA Ladder with the controllable size and concentration can be rapidly obtained with the low cost. The problems that a traditional enzyme digestion method is high in cost, and the size and concentration of the DNA fragments are not controlled are solved. The DNA Ladder obtained by adopting the method is basically equivalent to the commercial DNA Ladder in definition and indication effect, and can replace the commercial DNA Ladder for usage.
Description
Technical field
The present invention relates to plant genetic engineering field, especially a kind of fast and low-cost prepares the method for DNA Ladder.
Background technology
DNA ladder is the DNA fragmentation biased sample of one group of known dimensions and concentration, can carry out gel electrophoresis for just slightly indicating its molecular weight and concentration together with the DNA sample of the unknown, is one of reagent consumptive material that molecular biology experiment is conventional.
Early stage DNA ladder uses restriction enzyme (as ECOR I or Hind Ш etc.) enzyme cut the genomic dna of lambda particles phage or the plasmid DNA fragment of monkey virus and bacteria and obtain the DNA fragmentation varied in size.Human and material resources and the instrument maintenance cost of this method are high especially, and the restriction of the size producing DNA fragmentation being usually restricted property restriction enzyme site, can not the clip size of artificial regulatory DNA ladder and concentration.
PCR can fast and effeciently obtain a large amount of target fragment, and by the fragment of the acquisition arbitrary size of design of primers high efficiency low cost in suitable scope (100bp-3000bp), by being building up on pMD18-T carrier containing target sizes fragment, the universal primer on carrier is utilized to carry out pcr amplification to each fragment respectively, each DNA fragmentation of DNA ladder carries out diluting and mixing according to designing concentration again, the DNA ladder that final preparation size cheap fast, concentration are all controlled.
Summary of the invention
The object of the invention is: provide a kind of fast and low-cost to prepare the method for DNA Ladder, it solve traditional enzymatic cleavage methods cost high, obtain DNA fragmentation size and the uncontrollable difficult problem of concentration, to overcome the deficiencies in the prior art.
The present invention is achieved in that fast and low-cost prepares the method for DNA Ladder, according to the stripe size required for DNA Ladder, the DNA sequence dna of target sizes is chosen from known group sequence, adopt the primer designed to carry out PCR to increase, cut after glue reclaims and the band of different size is building up to pMD18-T carrier respectively, and extract plasmid, obtain the template of each fragment; Measure the concentration of the object fragment after each purifying, carry out mixing according to the concentration designed and dilute, obtaining final DNA ladder.
The amplified production of each DNA fragmentation is carried out purifying specifically: utilize universal primer M13F and the M13R on pMD18-T carrier, respectively each object fragment is increased, by desalting and purifying PCR primer, obtain each object fragment, utilize 10 × TE buffer solution, can preserve for a long time.
PCR can fast and effeciently obtain a large amount of target fragment, and by the fragment of the acquisition arbitrary size of design of primers high efficiency low cost in suitable scope (100bp-3000bp),
Owing to have employed above technical scheme, the present invention obtains the band required for DNA Ladder by the method for pcr amplification, the size of all bands and concentration can accurately control, not by the restriction of traditional method (as EcoR I enzyme cuts λ DNA) by restriction enzyme site and plasmid concentration, be produced into instinct and reduce by 90%; Again by being building up on pMD18-T carrier containing target sizes fragment, the universal primer on carrier is utilized to carry out pcr amplification to each fragment respectively, each DNA fragmentation of DNA ladder carries out diluting and mixing according to designing concentration again, the DNA ladder that final preparation size cheap fast, concentration are all controlled.Solve traditional enzymatic cleavage methods cost high, obtain DNA fragmentation size and the uncontrollable difficult problem of concentration.And the DNA ladder sharpness adopting the inventive method to manufacture is substantially suitable with commercial DNA ladder with indicating effect, commercial DNA ladder can be replaced to use.The inventive method is simple, and easy to implement, with low cost, result of use is good.
Accompanying drawing illustrates,
Fig. 1 is schematic flow sheet of the present invention;
Fig. 2 is the comparison diagram of preparation DNA ladder and commercial DNA ladder of the present invention.
Embodiment,
Embodiments of the invention: fast and low-cost prepares the method for DNA Ladder
The clone of step one, T100 ~ T3000 template and structure
In Tair database, the DNA sequence dna of appropriate length is selected in retrieval, design of amplification primers, with the tender colored cDNA of Arabidopis thaliana children for template carries out PCR amplification, 3000bp is cloned from arabidopsis gene group, 2000bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 300 bp, the DNA fragmentation of 100bp, combination of primers is followed successively by (DLF+3000R, DLF+2000R, DLF+1000R, DLF+750R, DLF+500R, DLF+300R, DLF+100R) its corresponding amplimer sequence is shown in sequence table, and be building up to pMD8-T carrier respectively, called after T-100 ~ T3000 respectively, pick out positive colony, and prepare positive plasmid with plasmid extraction kit (the raw work in Shanghai), concrete operations reference view 1, sequence information is shown in sequence table.
Step 2, T100 ~ T3000 rapid amplifying and purifying
Common primers M13F and M13R on pMD8-T carrier is adopted to increase to the carrying out of T100 ~ T3000 respectively, chloroform alcohol purification process is adopted to carry out desalting treatment, dissolve with 10 × TE buffer, obtain large, high each DNA object fragment that can preserve for a long time of purity of concentration.
(1) PCR reaction mixture (Shanghai raw work 2 × Taq PCR reaction kit) is configured:
2×Taq PCR Mixture 10μl
Forward primer M13 F(10 μM) 1 μ l
Reverse primer M13 R(10 μM) 1 μ l
Plasmid DNA (10ng/ μ l) 2 μ l
dd H
2O 6μl
The DNA fragmentation of each different size prepares 10 parts respectively.
(2) carry out PCR reaction, time and the temperature of reaction are as follows:
94℃ 3min
94℃ 30s，
58 DEG C of 30s ~ 3min(according to target fragment length, 1 Kb/min)
72℃ 30s，40cycles
72℃ 5min
(3) purifying and dissolving:
Collect together by the amplified production of each DNA fragmentation respectively, add 200 μ l chloroforms (traditional Chinese medicines), thermal agitation 30s, room temperature places 5min, 12000g, 4 DEG C, centrifugal 10min, gets in supernatant to new pipe, add 500 μ l dehydrated alcohols, after mixing, place 20min, 12000g, 4 DEG C for-20 DEG C, centrifugal 10min, removes supernatant, adds 1ml 75% ethanol, 10000g, 5min, 4 DEG C, centrifugal 10min, remove supernatant, dry air 15min, add 50 μ l 10 × TE buffer 50-60 DEG C and dissolve 5min; Get 1 μ l and dilute 10 times, measure concentration for subsequent use.
The mixing of step 3, DNA ladder and detection
According to the concentration required for DNA ladder, with each DNA fragmentation of 10 × TE buffer dilution mixture; Adding tetrabromophenol sulfonphthalein makes final concentration be 1g/L; Get 5 μ l electrophoresis together with commercial DNA ladder, as shown in Figure 2, the DNA ladder adopting the present invention to prepare is suitable with commercial DNA ladder effect.
Of the present inventionly be not limited to the embodiment described in embodiment, those skilled in the art's technical scheme according to the present invention draws and other embodiment belongs to technological innovation scope of the present invention equally.Obvious those skilled in the art can carry out various change and modification to the present invention and not depart from the spirit and scope of the present invention.Like this, if these amendments of the present invention and modification belong within the scope of the claims in the present invention and equivalent technologies thereof, then the present invention is also intended to comprise these change and modification.
SEQUENCE LISTING
sequence table
<110> Guizhou Province Grass Industry Research Institute
<120> fast and low-cost prepares the method for DNA Ladder
<130> nm:
<160> 11
<170> PatentIn version
<210> 1
<211>
<212> DNA
<213> artificial sequence
<220>
<223> is template according to the sequence of the IKU2 gene of TAIR database retrieval, designs forward primer DLF for each base slice sequence of cloned DNA ladder with Primer Premier 5.0.
<400> 1
ATGCT CCGGC TACTA TTTAT CGTTCG 26
<210> 2
<211> 28
<212> DNA
<213> artificial sequence
<220>
<223> is template according to the sequence of the IKU2 gene of TAIR database retrieval, design reverse primer 3000R with Primer Premier 5.0, match for the 3000bp base slice needed for cloned DNA ladder with DLF.
<400> 2
TAATA ACCAA GTGCA ATCTA TGAGT TAT 28
<210>2
<211> 24
<212> DNA
<213> artificial sequence
<220>
<223> is template according to the sequence of the IKU2 gene of TAIR database retrieval, design reverse primer 2000R with Primer Premier 5.0, match for the 2000bp base slice needed for cloned DNA ladder with DLF.
<400> 3
CTCCA GCCCT TTAGC AGCTC CTAA 24
<210> 4
<211> 26
<212> DNA
<213> artificial sequence
<220>
<223> is template according to the sequence of the IKU2 gene of TAIR database retrieval, design reverse primer 1500R with Primer Premier 5.0, match for the 1500bp base slice needed for cloned DNA ladder with DLF.
<400> 4
TATAG CACCA GATAG ATTAT TCTGG T 26
<210> 5
<211> 25
<212> DNA
<213> artificial sequence
<220>
<223> is template according to the sequence of the IKU2 gene of TAIR database retrieval, design reverse primer 1000R with Primer Premier 5.0, match for the 1000bp base slice needed for cloned DNA ladder with DLF.
<400> 5
TATAG CACCA GATAG ATTAT TCTGG 25
<210> 6
<211> 25
<212> DNA
<213> artificial sequence
<220>
<223> is template according to the sequence of the IKU2 gene of TAIR database retrieval, designs reverse primer 750R with Primer Premier 5.0, matches for the 750bp base slice needed for cloned DNA ladder with DLF.
<400> 6
AAGCT GCCTT AGGTT TTTGA GCTGA 25
<210> 7
<211> 24
<212> DNA
<213> artificial sequence
<220>
<223> is template according to the sequence of the IKU2 gene of TAIR database retrieval, designs reverse primer 500R with Primer Premier 5.0, matches for the 500bp base slice needed for cloned DNA ladder with DLF.
<400> 7
GAACT CCATG GGAAT ATTCC AGAG 24
<210> 8
<211> 23
<212> DNA
<213> artificial sequence
<220>
<223> is template according to the sequence of the IKU2 gene of TAIR database retrieval, designs reverse primer 300R with Primer Premier 5.0, matches for the 300bp base slice needed for cloned DNA ladder with DLF.
<400> 8
CTTCA AATCG CAGAT TGAAT CGA 23
<210> 9
<211> 26
<212> DNA
<213> artificial sequence
<220>
<223> is template according to the sequence of the IKU2 gene of TAIR database retrieval, designs reverse primer 100R with Primer Premier 5.0, matches for the 100bp base slice needed for cloned DNA ladder with DLF.
<400> 9
TCAGT TTGAG AAGAT TCTCG ACTTC T 26
<210> 10
<211> 24
<212> DNA
<213> artificial sequence
<220>
The pMD18-T sequence that <223> retrieves according to ncbi database, designs forward primer M13F with Primer Premier 5.0, matches be used for the object fragment of a large amount of DNA amplification ladder with M13R.
<400> 10
CGCCA GGGTT TTCCC AGTCA CGAC
<210> 11
<211> 24
<212> DNA
<213> artificial sequence
<220>
The pMD18-T sequence that <223> retrieves according to ncbi database, designs forward primer M13R with Primer Premier 5.0, matches be used for the object fragment of a large amount of DNA amplification ladder with M13F.
<400> 11
GAGCG GATAA CAATT TCACA CAGG
Claims (2)
1. a fast and low-cost prepares the method for DNA Ladder, it is characterized in that: according to the stripe size required for DNA Ladder, the DNA sequence dna of target sizes is chosen from known group sequence, adopt the primer designed to carry out PCR to increase, cut after glue reclaims and the band of different size is building up to pMD18-T carrier respectively, and extract plasmid, obtain the template of each fragment; Adopt common primers on carrier to increase in a large number and obtain an object fragment, measure its concentration, carry out mixing according to the concentration designed and dilute, obtaining final DNA ladder.
2. fast and low-cost according to claim 1 prepares the method for DNA Ladder, it is characterized in that: the amplified production of each DNA fragmentation is carried out purifying specifically: utilize universal primer M13F and the M13R on pMD18-T carrier, respectively each object fragment is increased, by desalting and purifying PCR primer, obtain each object fragment, utilize 10 × TE buffer solution.
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| CN201510265744.0A CN104988134A (en) | 2015-05-24 | 2015-05-24 | Method for rapidly preparing DNA Ladder with low cost |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109136218A (en) * | 2018-08-28 | 2019-01-04 | 大连民族大学 | Paeonia papaveracea IKU2 gene preparation method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004063322A2 (en) * | 2003-01-13 | 2004-07-29 | Seegene, Inc. | Dna size markers and method for preparing them |
| CN102304508A (en) * | 2011-07-29 | 2012-01-04 | 上海捷瑞生物工程有限公司 | Method for preparing DL2000 DNA molecular weight marker as well as product and applications thereof |
| CN103667264A (en) * | 2012-08-31 | 2014-03-26 | 沙船（天津）生物科技发展有限公司 | Preparation method of deoxyribonucleic acid (DNA) Marker |
-
2015
- 2015-05-24 CN CN201510265744.0A patent/CN104988134A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004063322A2 (en) * | 2003-01-13 | 2004-07-29 | Seegene, Inc. | Dna size markers and method for preparing them |
| CN102304508A (en) * | 2011-07-29 | 2012-01-04 | 上海捷瑞生物工程有限公司 | Method for preparing DL2000 DNA molecular weight marker as well as product and applications thereof |
| CN103667264A (en) * | 2012-08-31 | 2014-03-26 | 沙船（天津）生物科技发展有限公司 | Preparation method of deoxyribonucleic acid (DNA) Marker |
Non-Patent Citations (2)
| Title |
|---|
| 刘慧娟等: "利用PCR技术快速制备DNA分子量标准物", 《生物技术》 * |
| 王俐等: "DNA Ladder的制备", 《中国组织工程研究与临床康复》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109136218A (en) * | 2018-08-28 | 2019-01-04 | 大连民族大学 | Paeonia papaveracea IKU2 gene preparation method |
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Application publication date: 20151021 |