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A PCR-independent short-fragment cloning method

A cloning method and short fragment technology, applied in the field of bioengineering, can solve the problems of needing templates, long time-consuming, complicated and time-consuming operations, etc.

Active Publication Date: 2022-07-08
HENAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main disadvantages of this method are: cumbersome operation and long time-consuming: templates are required and after PCR amplification, operations such as gel electrophoresis identification, purification and recovery are required
Although this connection method can guarantee the connection length, the operation is complicated and time-consuming

Method used

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  • A PCR-independent short-fragment cloning method
  • A PCR-independent short-fragment cloning method
  • A PCR-independent short-fragment cloning method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Take the restriction endonuclease site adjustment in the multiple cloning site in the gene silencing vector pTRV2 for virus induction as an example (for the specific operation process, please refer to figure 1 ), in this example, the SalI restriction site was introduced, and the arrangement order of KpnI and BamHI in the multiple cloning site was adjusted. The specific experimental process is introduced as follows.

[0070] (1) Preparation of linearized vector

[0071] The pTRV2 vector was linearized with EcoRI and XhoI, and purified and recovered. The specific operation reference is as follows:

[0072] The 50 μL digestion system was designed as follows:

[0073] pTRV2 vector, 2000 ng;

[0074] EcoRI, 2 μL;

[0075] XhoI, 2 μL;

[0076] 10x Cut smart buffer, 5 μL;

[0077] ddH 2 O, make up to 50 μL.

[0078] Digestion at 37°C overnight (about 10h), followed by purification and recovery of the digested linear vector product for later use.

[0079] (2) Design ...

Embodiment 2

[0105] Taking the binary expression vector pRI101-AN as an example, in this example, a Myc protein tag was added to its multi-cloning site region, so that the C-terminal of the protein expressed by the subsequent transgenic plants had this tag, which was beneficial to the subsequent protein expression. Level detection (for specific operation procedures, please refer to figure 2 ).

[0106] The specific experimental process is introduced as follows.

[0107] (1) Preparation of linearized vector

[0108] The pRI101-AN vector was linearized with NdeI and BamHI, and purified and recovered. The specific operation reference is as follows:

[0109] The 50 μL digestion system is as follows:

[0110] pRI101-AN vector, 2000 ng;

[0111] NdeI, 2 μL;

[0112] BamHI, 2 μL;

[0113] 10x Cut smart buffer, 5 μL;

[0114] ddH 2 O, make up to 50 μL

[0115] Digestion at 37°C overnight (about 10h), followed by purification and recovery of the digested linear vector product for later u...

Embodiment 3

[0140] Taking the binary expression vector pRI101-AN as an example, in this example, two protein tags, Myc and His, were added to its multi-cloning site region, so that the protein C-terminus expressed by the subsequent transgenic plants had this tag, which was beneficial to the Subsequent detection of protein expression levels (for specific operation procedures, please refer to image 3 ).

[0141] (1) Preparation of linearized vector

[0142] The pRI101-AN vector was linearized with NdeI and BamHI, and purified and recovered. The specific operation reference is as follows:

[0143] The 50 μL digestion system is as follows:

[0144] pRI101-AN vector, 2000 ng;

[0145] NdeI, 2 μL;

[0146] BamHI, 2 μL;

[0147] 10x Cut smart buffer, 5 μL;

[0148] ddH 2 O, make up to 50 μL;

[0149] Digestion at 37°C overnight (about 10h), followed by purification and recovery of the digested linear vector product for later use.

[0150] (2) Design and synthesis of two long-chain oli...

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Abstract

The present application belongs to the technical field of bioengineering, and in particular relates to a method for rapid cloning of short fragments that does not rely on PCR reaction. The method includes the steps of linearizing the vector, designing and synthesizing two partially complementary long-chain oligonucleotide sequences, preparing the target sequence, homologous recombination, transformation, screening, and obtaining the recombinant vector. In this application, the inventor has further improved the preparation method of the target fragment in the existing recombination reaction, using the single-strand recombination method to extend the length of the inserted sequence, and using the natural repair system of Escherichia coli to complete the double-strand completion, thereby To achieve the purpose of rapid cloning of short fragments. The invention retains the simple and fast technical characteristics of the existing recombination method, and simultaneously has the technical advantages of the existing connection method in terms of connection length. It can lay a good technical foundation for recombinant plasmid transformation (such as enzyme cutting site transformation, addition of protein expression tags, etc.).

Description

technical field [0001] The present application belongs to the technical field of bioengineering, and in particular relates to a method for rapid cloning of short fragments that does not rely on PCR reaction. Background technique [0002] Gene cloning is the main technical means of gene research, and usually requires the help of different types of vectors to achieve the cloning and expression of target genes. In practice, the vector usually needs to be modified to meet the needs of the experiment, such as the replacement of polyclonal restriction sites and the addition or removal of expression tags. These transformations often only require replacing very short fragments. [0003] In the prior art, gene cloning for short fragments (generally referred to as fragments below 100 bp) can be divided into the following two categories according to whether the PCR reaction is relied upon. [0004] The method relying on PCR reaction: this method is the same as the strategy of long fr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12N15/70
CPCC12N15/10C12N15/70Y02A50/30
Inventor 何世斌王翔伍郝云飞王鲜萍张鹏辉吉逢逢
Owner HENAN UNIVERSITY
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