Directional cloning method, transformation method of carrier and carrier T

A vector and directional technology, applied in the field of bioengineering, can solve the problem that T vector cannot be directional cloning, and achieve the effect of saving research time, high application value and improving success rate.

Inactive Publication Date: 2013-04-03
SHENZHEN UNIV
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a method for directional cloning and a transformation method of a carrier and a T carrier, and utilize a variable restriction endonuclease Xcm I to prepare a T carrier capable of directional cloning efficiently and quickly. , aiming to solve the problem that existing T vectors cannot perform directional cloning

Method used

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  • Directional cloning method, transformation method of carrier and carrier T
  • Directional cloning method, transformation method of carrier and carrier T
  • Directional cloning method, transformation method of carrier and carrier T

Examples

Experimental program
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Effect test

Embodiment 1

[0068] Example 1: Construction of prokaryotic expression T-vector

[0069] By transforming the prokaryotic expression vector pQE-30a, a restriction cassette containing the kanamycin resistance gene with Xcm I endonuclease cut sites on both ends was designed, and other restriction cuts were added to both ends of the restriction cassette. Site, and then directional clone the restriction cassette containing the kanamycin resistance gene with Xcm I endonuclease sites at both ends by recombination technology to the corresponding restriction site on the pQE-30a vector, and the sequencing will be correct The recombinant plasmid was then cut with Xcm I endonuclease to cut the fragment with kanamycin resistance gene to form a linearized T-vector with protruding dT at both ends. Finally, human retinol binding protein (hRBP) was used to identify the expression function of the constructed T vector. After PCR, the DNA fragment of hRBP gene was ligated with T4 ligase and linearized T vector, a...

Embodiment 2

[0188] Example 2 Construction of yeast surface display T vector and its function identification

[0189] The sequence of the yeast surface display T vector pYD-T vector restriction cassette constructed in this example is as follows Figure 16 As shown, a restriction cassette with Xcm I endonuclease cutting sites at both ends was designed, and NheI, BamH I, NdeI, and Xho I restriction sites were added to both ends of the restriction cassette, and then recombined into Nhe I 、On the pYD-1 vector after XholI digestion treatment, cut the correctly sequenced recombinant plasmid with Xcm I endonuclease, and cut the fragment containing the yellow fluorescent protein gene to form two ends with Linearized T-carrier with prominent dT. Finally, the red fluorescent protein (RFP) was used to identify the expression function of the constructed T vector. The DNA fragment of the RFP gene was amplified by PCR and then ligated with the linearized T vector with T4 ligase, and then introduced into Sa...

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Abstract

The invention discloses a directional cloning method, a transformation method of a carrier and a carrier T, wherein the directional cloning method includes the steps of designing and synthesizing an enzyme digestion kit, constructing the linear carrier T with two ends provided with protruded dT, generating PCR (Polymerase Chain Reaction) segment of the target gene, and constructing a recombinant carrier containing the target gene. According to the directional cloning method, the directional TA cloning technology can be used for preparing efficient directionally-cloned carrier T which can be widely applied to cloning and expression of a PCR product in molecular biology experiment. As an expression carrier is reconstructed, the PCR product can be directly connected to the expression carrier, the connecting direction of the target gene can be determined after identification of single enzyme digestion, expression can be performed in cells, the connecting direction is determined without complex steps such as conventional sequencing and the like, the direction cloning can be simply and rapidly realized, engineering bacteria can be constructed through one step, and the application prospect is wide.

Description

Technical field [0001] The invention relates to the field of bioengineering, in particular to a directional cloning method, a vector transformation method and a T vector. Background technique [0002] PCR is currently the most commonly used method for in vitro gene amplification. The rapid and effective cloning of PCR products is an effective method for gene preservation, amplification, sequencing and expression. For this reason, many scholars at home and abroad have explored many different cloning methods. Commonly used methods include non-directional cloning and directional cloning. Both methods have established corresponding vectors. Although these existing cloning vectors can achieve the purpose of cloning, they also have certain shortcomings. [0003] The obvious advantage of T vector for rapid and efficient cloning of PCR products shows its application prospects in the construction of various gene libraries. Since the 3'end of the vector has a prominent 3'dT residue, it can ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/66C12N15/63
Inventor 田生礼梁志成梁秀怡刘士德
Owner SHENZHEN UNIV
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