Temperature-controlled zero background T-vector precursor and construction method and application thereof

A zero-background, temperature-controlled technology, applied in the field of genetic engineering, can solve problems such as large volume, unfavorable large fragment cloning, and reduced vector cloning transformation efficiency, achieving the effect of reducing costs

Inactive Publication Date: 2014-01-08
YANGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, compared with commercial T vectors usually less than 3000bp in size, the volume of the T vector (3929bp) is too large, which is not conducive to the cloning of large fra

Method used

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  • Temperature-controlled zero background T-vector precursor and construction method and application thereof
  • Temperature-controlled zero background T-vector precursor and construction method and application thereof
  • Temperature-controlled zero background T-vector precursor and construction method and application thereof

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preparation example Construction

[0042] 2. Preparation of T vector (XcmI enzyme digestion method)

[0043] 2.1 Pre-T vector preparation

[0044] Inoculate Escherichia coli DH5α strain carrying pre-T vector pFLX107 into LB liquid medium containing 34 μg / mL chloramphenicol, and shake overnight at 28°C on a shaker. Plasmids were extracted using the High-Speed ​​Plasmid Mini Kit (Geneaid) kit according to the manufacturer's instructions.

[0045] 2.2XcmI digestion

[0046] Digest the pre-T vector obtained in 2.1. As mentioned above, the pre-T vector contains two XcmI restriction sites, and each restriction site will leave a band at the end of the final linearized vector 3'-dT overhang. XcmI was purchased from NEB (Nanjing) Co., Ltd., and the enzyme digestion system was as follows:

[0047]

[0048] The reaction conditions are: 37°C, 3h.

[0049] After the enzyme digestion reaction, agarose gel electrophoresis was used to detect whether the enzyme digestion was complete.

[0050] Recovery and purification...

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Abstract

The invention relates to a temperature-controlled zero background T-vector precursor and a construction method and application thereof. The temperature-controlled zero background t-vector precursor is a vector pFLX107, and a sequence of the same is shown in SEQ ID NO.1. Volume of the successfully constructed vector is 3029bp, volume of a T-vector prepared via Xcml enzyme digestion is only 2747bp and approximate to those of various commercial T-vectors in the market at present, and the T-vector can be widely applied to TA cloning of various exogenous PCR (polymerase chain reaction) fragments. Due to presence of temperature controlled lethal gene, positive clone selection of recombined transformants by 100% can be achieved at the temperature of 37 DEG C. Meanwhile, since universal sequencing primer sequence binding sites which can be provided by biological companies are integrated on two sides of a cloning site in the vector, primers are not required to be provided by oneself during sample presentation and sequencing, and cost is further lowered.

Description

technical field [0001] The invention relates to the construction and preparation of a carrier in the field of genetic engineering, in particular to a temperature-controlled zero-background T carrier precursor and its construction method and application. Background technique [0002] T vector is the core tool of TA cloning, the most commonly used molecular cloning technique in molecular biology experiments. Currently, there are two main methods for the preparation of T vectors. The first method adds a single deoxythymonucleotide (dTTP) to the 3' end of a linearized vector digested with a blunt end restriction enzyme by terminal transferase or Taq (Holton and Graham, 1991); The second method is to introduce specific restriction enzyme sites arranged in tandem into the vector, such as AspEI, Eam1105I or XcmI, etc., and then digest with the corresponding endonuclease to generate a linear T with a single T overhang at the 3' end. Vector (Ichihara and Kurosawa, 1993; Harrison et...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/64
Inventor 付立霞魏文志王秀英
Owner YANGZHOU UNIV
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