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Cloning vector

A carrier and drug resistance technology, which is applied in the field of PCR product cloning vectors, can solve the problem of not being able to control the directionality of the target gene, and achieve the effect of simplifying the preparation operation.

Active Publication Date: 2012-03-21
TAKARA HOLDINGS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in this method, the directionality of the gene of interest inserted into the vector cannot be controlled

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0077] (1) Construction of the vector

[0078] The vector of the present invention is constructed by the following method. First, the pUC19 vector (manufactured by Takara Bio Co., Ltd.) was digested with restriction enzymes Nde I and PvuII (both manufactured by Takara Bio Co., Ltd.). This was followed by blunt endization and BAP treatment. The DNA fragment thus obtained is purified by a conventional method. Next, a double-stranded DNA having a nucleic acid sequence from the 999th base to the 213th base in the nucleic acid sequence described in SEQ ID NO: 5 of the Sequence Listing is synthesized. Next, this double-stranded DNA was inserted into pUC 19 DNA cut with restriction enzymes Nde I and Pvu II. The plasmid DNA thus constructed was named pUC19-Kana DNA.

[0079] Inverse PCR was performed using the above pUC19-Kana DNA as a template. First, AMP-F primers and AMP-R primers each having the nucleotide sequences described in SEQ ID NOs: 3 and 4 of the Sequence Listing wer...

Embodiment 2

[0084] Example 2 Regarding the pUC19-blaΔW290-T DNA prepared in Example 1, PCR fragments of various chain lengths were inserted by the TA cloning method to discuss the efficiency of obtaining positive clones.

[0085] (1) Discussion on the efficiency of obtaining positive clones of 500bp PCR fragments

[0086] A 500bp-F primer and a 500bp-R primer having the nucleic acid sequences described in SEQ ID NOs: 6 and 7 of the Sequence Listing, respectively, were synthesized. Next, PCR was performed using these primers using Escherichia coli JM 109 genomic DNA as a template. PCR was performed in the following reaction composition. That is, 50 μL of the reaction solution contained 25 μL of 2×Ex Taq Premix (manufactured by Takara Bio Co., Ltd.), 20 pmol of each of the aforementioned primers, and 50 ng of template DNA. The PCR conditions were 1 minute at 94°C, (15 seconds at 98°C, 30 seconds at 59°C, and 30 seconds at 72°C) as one cycle, followed by 30 cycles of reaction, followed by ...

Embodiment 3

[0097] The relationship between the vectors and inserts of the present invention is discussed. In this example, the 500 bp PCR amplified fragment and the 2 kbp PCR amplified fragment prepared in the above-mentioned Example 2(1) and Example 2(3) were used. Six kinds of 10 μL reaction solutions were prepared, which respectively contained 25 ng, 50 ng or 100 ng of the vector of the present invention relative to 100 ng of the 500 bp or 2 kbp amplified fragment, and DNA Ligation kit ver. These solutions were kept at 16°C for 1 hour. Transformation and culture conditions were the same as in Example 2(1). As a result, in the case of inserting a 500 bp PCR fragment, it was confirmed that the number of clones obtained was increased as the vector concentration increased. On the other hand, when the 2 kbp PCR fragment was inserted, it was confirmed that the maximum number of clones was obtained when the vector amount was 50 ng.

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Abstract

The present invention provides a TA cloning vector, a cloning method using the vector, a manufacturing method for the vector and a kit containing the vector, wherein the TA cloning vector controls the insertion direction of the DNA fragment of PCR augmentation products, and simultaneously can easily select and insets the purposed DNA garments for cloning. The present invention provides a vector, wherein the tail end of the vector is provided with an activity-lost drug-fast gene with the nucleic acid sequence deficient, and the nucleic acid sequence codes the amino acid of the C tail end portion obbligato in the express process of the drug-fast performance of the drug-fast gene products and the amino acid of the N tail end portion. The present invention also provides a cloning method using the vector, the manufacturing method for the vector and a kit containing the vector.

Description

technical field [0001] The present invention relates to a vector suitable for cloning of PCR products, a cloning method using the vector, a method for producing the vector, and a kit containing the vector. Background technique [0002] Heretofore, a method using PCR has been known as a method for isolating a gene of interest. In this method, DNA containing the gene of interest or a fragment thereof is first amplified by PCR, the resulting PCR product is assembled into a plasmid vector for cloning, and the plasmid vector is further used to transform the host to form a single colony of transformants. Next, the plasmid was purified from the formed single colony, and the DNA fragment inserted into the plasmid was confirmed. By doing so, a plasmid into which the gene of interest or a fragment thereof is inserted can be obtained. [0003] Among the enzymes used in the PCR method, the typical Taq DNA polymerase is known to have a side reaction activity of adding one deoxyadenosin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/66C12N15/64
Inventor 棚濑智雄
Owner TAKARA HOLDINGS
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