CN107058357A - A kind of TA cloning vectors and its construction method and application based on digestion - Google Patents

Abstract

The invention discloses a kind of TA cloning vectors based on digestion and its construction method and application, the nucleotide sequence of the TA cloning vectors based on digestion is named as pTA03 carriers as shown in SEQ ID NO.1.After the carrier is digested through restriction enzyme A hdI, the linear carrier fragment of purifying can be used for TA to clone, and the carrier carries ccdB lethal genes, be screened without carrying out blue hickie, and positive rate is close to 100%.Program reliability is high, and cost is relatively low, can be prepared on a large scale.

Description

A kind of TA cloning vector based on enzyme cutting and its construction method and application

technical field

The invention belongs to the field of molecular biology, and in particular relates to a TA cloning vector based on enzyme cutting and its construction method and application.

Background technique

TA cloning is currently the most convenient, fast and economical method for cloning PCR products, and it is widely used in the field of molecular biology. According to the types of enzymes used in the reaction process, TA cloning can be mainly divided into two categories, one is based on T4 DNA ligase, and the other is based on DNA topoisomerase I (TOPO). The former vector is easy to prepare and low in cost. High-efficiency connection takes a long time, the preparation of the latter carrier is cumbersome and costly, and high-efficiency connection takes a very short time. According to the method of generating T-terminus of the vector, there are two ways to use transferase to add T to the end of the PCR product and to generate T-terminus by enzymatic digestion. The former has high throughput and poor consistency, while the latter has slightly lower flux and good consistency.

Contents of the invention

In view of this, the present invention aims at the above problems, and provides a TA cloning vector based on enzyme cutting and its construction method and application. The TA cloning vector has high reliability, good consistency, and low cost, and can be used in small and medium-sized laboratories. use.

In order to solve the above technical problems, the present invention discloses a TA cloning vector based on enzyme cutting, the nucleotide sequence of the TA cloning vector based on enzyme cutting is shown in SEQ ID NO.1, named as pTA03 vector.

The present invention also provides a method for constructing a TA cloning vector based on enzyme digestion, comprising the following steps: take 3-5 μg of the pTA03 vector, digest it with the restriction endonuclease AhdI for 3-18 hours at 37°C, and Add a small amount of EcoRV-HF endonuclease to digest for 30 minutes, keep at 65°C for 20 minutes, and use the enzyme digestion kit to directly pass through the column to recover the digested product, or use the DNA gel recovery kit to recover; the recovered product should be stored at -20°C. The product is the prepared TA cloning vector.

The present invention also provides a method for using the above enzyme-based TA cloning vector, comprising the following steps:

Step 1. Preparation of TA cloning vector based on enzyme digestion: take 3-5 μg of pTA03 vector, digest it with restriction endonuclease AhdI for 3-18 hours at 37°C, and add a small amount of EcoRV-HF endogenous digestion after enzyme digestion Enzyme digested for 30 minutes, kept at 65°C for 20 minutes, and the digested product was recovered directly through the column with an enzyme digestion kit, or recovered with a DNA gel recovery kit; the recovered product was stored at -20°C, and the product was the prepared TA clone carrier;

Step 2. Mix the TA cloning vector based on enzyme digestion prepared in step 1 and the target PCR fragment to prepare a ligation reaction system. After mixing, centrifuge briefly for 3-5 seconds, and react the mixture at 22 degrees for 5 minutes. On ice, carry out subsequent transformation reactions.

Further, the enzyme digestion reaction system in step 1 is specifically: 10 μL of 10xCutSmart Buffer (NEB), 2 μL of restriction endonuclease AhdI of 10 U/μL, 5 μL of TA cloning vector based on enzyme digestion of 1 μg/μl, and the balance is H ₂ O, the total volume of the above is 100 μL.

Further, the ligation reaction system in step 2 is specifically: 1 μL of 10xT4DNA ligase Buffer (NEB), 0.5 μL of 400 U/μL T4DNA ligase, 1 μL of TA cloning vector based on enzyme digestion at 50 ng/μL, and the target PCR fragment and enzyme-based digestion The molar ratio of the TA cloning vector is 3-7:1, the balance is ddH ₂ O, and the total volume of the above is 100 μL.

Further, the addition amount of the TA cloning vector based on enzyme cutting in the ligation system is 0.03pmol, and when the length of the target PCR fragment is below 2000bp, the molar ratio of the target PCR fragment: the TA cloning vector based on enzyme cutting is 7:1 , ligated for 5 minutes at 22°C; when the length of the target PCR fragment was more than 2000bp, the molar ratio of the target PCR fragment: TA cloning vector was 3:1, ligated for 30 minutes at 22°C.

Further, the amount of the target PCR fragment (μl)=[660×A×0.03×B(ng)]÷[1000×the concentration of the target PCR fragment (ng/μl)], where A is the number of base pairs of the target PCR fragment (bp), B is the molar ratio of PCR fragment: TA cloning vector.

Compared with prior art, the present invention can obtain and comprise following technical effect:

1) The TA cloning vector of the present invention is simple to prepare and low in cost, and a large number of vectors can be prepared by one enzyme digestion;

2) The TA cloning vector of the present invention has high reliability and good consistency, and basically all ends have T;

3) The TA cloning vector of the present invention carries a lethal gene, which does not require blue-white screening, and the positive rate is close to 100%.

Of course, implementing any product of the present invention does not necessarily need to achieve all the technical effects described above at the same time.

Description of drawings

The accompanying drawings described here are used to provide a further understanding of the present invention, and constitute a part of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention, and do not constitute improper limitations to the present invention. In the attached picture:

Fig. 1 is the schematic diagram of the TA cloning vector based on enzyme cutting of the present invention;

Fig. 2 is the growth situation of positive bacteria of the present invention on LB/Amp plate culture medium;

Fig. 3 is the result of PCR identification of different colonies of the present invention, wherein, M is D2000 DNA Marker, and 1-6 are random PCR identifications.

detailed description

The implementation of the present invention will be described in detail below with examples, so as to fully understand and implement the implementation process of how the present invention uses technical means to solve technical problems and achieve technical effects.

Embodiment 1 Restriction-based TA cloning vector and construction method thereof

The present invention provides a TA cloning vector based on enzyme cutting, which is constructed from pUC19 vector as the backbone, does not contain multiple cloning sites, carries ccdB lethal gene, has a positive rate close to 100%, and is suitable for DNA sequencing. After the vector was digested with the restriction endonuclease AhdI, the linearized TA cloning vector was obtained after purification and recovery, which was named pTA03 vector. The vector map is shown in Figure 1, the nucleotide sequence of the TA cloning vector is shown in SEQ ID NO.1, and its target fragment sequence is as follows: wherein are the M13fwd and M13rev primer sequences, respectively, is the AmpR sequence, It is the restriction endonuclease AhdI sequence, and the bold part is the ccdB lethal gene fragment.

The nucleotide sequence of the TA cloning vector is as follows:

Embodiment 2 The construction and use method of the TA cloning vector based on enzyme cutting

Take 3-5μg pTA03 plasmid, digest with restriction endonuclease AhdI for 3-18 hours, add a small amount of EcoRV-HF endonuclease to digest for 30 minutes, keep at 65°C for 20 minutes, and digest the product with enzyme The cutting kit can be directly recovered through the column, and the DNA gel recovery kit can also be used for recovery. The recovered product is stored at -20°C. This product is the prepared TA cloning vector. T4 DNA ligase can be used to perform TA ligation with the PCR product, and the ligated product does not need to be screened by blue and white spots.

Example 3

1. Vector linearization: As shown in Table 1, prepare enzyme digestion system, react at 37°C for 3-18 hours, add 1 μl EcoRV-HF endonuclease (NEB) to digest for 30 minutes after enzyme digestion, and keep at 65°C for 20 minutes , the digested product can be recovered directly through the column with the enzyme digestion kit, or can be recovered with the DNA gel recovery kit, and the recovered product can be stored at -20°C. Make a TA connection.

Table 1 enzyme digestion reaction system

2. TA connection: As shown in Table 2, prepare the connection reaction system according to the molar ratio of the target PCR fragment: TA cloning carrier at 3-7:1, mix well and centrifuge briefly for 3-5 seconds, and react the mixture at 22°C After 5 minutes, put it on ice after the reaction, and carry out the subsequent transformation reaction.

Table 2 Connection reaction system

Note: (1) The TA cloning carrier based on enzyme digestion added in the ligation system is 0.03pmol. When the length of the target PCR fragment is less than 2000bp, it is recommended that the molar ratio of the target PCR fragment: TA cloning carrier be 7:1, and ligate at 22°C 5 minutes; when the length of the target PCR fragment is more than 2000bp, it is recommended that the molar ratio of the target PCR fragment:TA cloning vector is 3:1, and ligate at 22°C for 30 minutes;

(2) The amount of the target PCR fragment (μl)=[660×A×0.03×B(ng)]÷[1000×the concentration of the target PCR fragment (ng/μl)], where A is the number of base pairs of the target PCR fragment (bp), B is the molar ratio of PCR fragment: TA cloning vector.

(3) The transformed bacterial strain should try to choose one that does not contain the laqI gene, such as DH5a, TOP10, T1, etc. If you need to use the JM109 strain, you should add a small amount of IPTG to the plate medium.

Example 4

As shown in Table 3, the ligation reaction system was prepared according to the target PCR fragment (about 150bp): TA cloning vector molar ratio of 7:1, the target PCR fragment is the FaMYB5 gene, its sequence is shown in SEQ ID NO.2, mixed After homogenization, briefly centrifuge for 3-5 seconds, react the mixture at 22°C for 5 minutes, put it on ice after the reaction, and take all the products to transform into DH5a. Figure 2 shows the growth of positive bacteria on the LB/Amp plate medium, randomly picked 6 colonies, and used M13fwd and M13rev primers to carry out colony PCR identification, as shown in Figure 3, the results showed that the amplified bands were all in line with expectations The sizes (about 500bp) are exactly the same, and further sequencing shows that the insertion of the target fragments is completely correct, which also confirms that the positive rate of the TA recombinant vector obtained by the present invention is close to 100%.

Table 3 Connection reaction system

The above description shows and describes several preferred embodiments of the invention, but as previously stated, it should be understood that the invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other embodiments. Combinations, modifications and circumstances, and can be modified within the scope of the inventive concept described herein, by the above teachings or by skill or knowledge in the relevant field. However, changes and changes made by those skilled in the art do not depart from the spirit and scope of the invention, and should be within the protection scope of the appended claims of the invention.

SEQUENCE LISTING

<110> Sichuan Agricultural University

<120> A TA Cloning Vector Based on Enzyme Cutting and Its Construction Method and Application

<130> 2017

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 2721

<212>DNA

<213> Synthetic

<400> 1

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccggggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaagtaaa acgacggcca gtttatattc cccagaacat 180

caggttaatg gcgtttttga tgtcattttc gcggtggctg agatcagcca cttcttcccc 240

gataacggag accggcacac tggccatatc ggtggtcatc atgcgccagc tttcatcccc 300

gatatgcacc accgggtaaa gttcacggga gactttatct gacagtcagt cgatatcgat 360

atcgatatcg atatcgacag acagtcgtgc actggccagg gggatcacca tccgtcgccc 420

cggcgtgtca ataatatcac tctgtacatc cacaaacaga cgataacggc tctctctttt 480

ataggtgtaa accttaaact gcatagctgt ttcctgtgtg aaattgttat ccgctcacaa 540

ttccacacat tatacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga 600

gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt 660

gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct 720

cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 780

cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 840

acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 900

ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 960

ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 1020

gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 1080

gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 1140

ccaagctggg ctgtgtgcac gaacccccccg ttcagcccga ccgctgcgcc ttatccggta 1200

actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 1260

gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 1320

ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 1380

ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 1440

gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 1500

tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 1560

tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 1620

aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 1680

aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctgg ctccccgtcg 1740

tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 1800

gagaccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 1860

agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 1920

aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 1980

gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 2040

caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 2100

cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 2160

ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 2220

ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 2280

gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 2340

cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 2400

gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 2460

caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 2520

tactcttcct ttttcaatat tattgaagca tttatcaggg ttaattgtctc atgagcggat 2580

acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 2640

aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc 2700

gtatcacgag gccctttcgt c 2721

<210> 2

<211> 139

<212>DNA

<213> FaMYB5 gene

<400> 2

tcctcggcaa tagatggtcc ctgattgctg ggaggattcc ggggcgaacg gacaatgaga 60

taaagaacta ctggaacact cacctcagta agaagctgat tagtcagggc atagatccca 120

gaacccacaa gccactcaa 139

Claims (7)

1. a kind of TA cloning vectors based on digestion, it is characterised in that the nucleotides sequence of the TA cloning vectors based on digestion Row are named as pTA03 carriers as shown in SEQ ID NO.1.

2. a kind of construction method of the TA cloning vectors as claimed in claim 1 based on digestion, it is characterised in that including following Step：Take under the conditions of 3-5 μ g pTA03 carriers, 37 DEG C and to be digested 3-18 hours with restriction enzyme A hdI, digestion terminates After add a small amount of EcoRV-HF endonuclease digestions 30 minutes, 65 DEG C keep 20 minutes, digestion products using cleavage reagent box it is direct Post recovery is crossed, or is reclaimed with DNA gel QIAquick Gel Extraction Kit；- 20 DEG C of preservations of recovery product, the product is TA gram prepared Grand carrier.

3. a kind of application method of the TA cloning vectors as claimed in claim 1 based on digestion, it is characterised in that including following Step：

Step 1, TA cloning vector of the preparation based on digestion：Take and use restriction enzyme under the conditions of 3-5 μ g pTA03 carriers, 37 DEG C AhdI is digested 3-18 hours, and digestion adds a small amount of EcoRV-HF endonuclease digestions 30 minutes after terminating, and 65 DEG C are kept for 20 points Clock, digestion products are directly crossed post using cleavage reagent box and reclaimed, or are reclaimed with DNA gel QIAquick Gel Extraction Kit；Recovery product -20 DEG C preserve, the product is the TA cloning vectors prepared；

Step 2, the TA cloning vectors and purpose PCR fragment mixed preparing coupled reaction body based on digestion for preparing step 1 System, of short duration centrifugation 3-5 seconds after mixing react mixed liquor 5 minutes in 22 degree, and reaction end is rearmounted on ice, carry out follow-up turn Change reaction.

4. the application method of the TA cloning vectors according to claim 3 based on digestion, it is characterised in that in step 1 Endonuclease reaction system is specially：μ L, the 10U/ μ L of 10xCutSmart Buffer (NEB) 10 restriction enzyme A hdI 2 μ L, 1 The μ g/ μ l μ L of TA cloning vectors 5 based on digestion, surplus is H₂O, using upper volume total amount as 100 μ L.

5. the application method of the TA cloning vectors according to claim 3 based on digestion, it is characterised in that in step 2 Coupled reaction system is specially：μ L, the 400U/ μ L of 10xT4 DNA ligase Buffer (NEB) 1 T4 DNA ligase 0.5 The mol ratio of μ L, the 50ng/ μ L μ L of TA cloning vectors 1, purpose PCR fragment and TA cloning vectors is 3~7：1, surplus is ddH₂O, using upper volume total amount as 100 μ L.

6. the application method of the TA cloning vectors according to claim 5 based on digestion, it is characterised in that in linked system The TA cloning vectors based on digestion addition be 0.03pmol, when the length of purpose PCR fragment is in below 2000bp, mesh PCR fragment：The mol ratio of TA cloning vectors is 7：1,22 DEG C connects 5 minutes；When purpose PCR fragment length 2000bp with When upper, purpose PCR fragment：The mol ratio of TA cloning vectors is 3：1,22 DEG C connects 30 minutes.

7. the application method of the TA cloning vectors according to claim 5 based on digestion, it is characterised in that purpose PCR pieces Consumption (μ l)=[660 × A × 0.03 × B (ng)] ÷ [1000 × purpose PCR fragment concentration (ng/ μ l)] of section, wherein A is mesh PCR fragment base logarithm (bp), B is PCR fragment：The mol ratio of TA cloning vectors.