Crispr/cas9 plasmid and its construction method and usage method

A plasmid and protoplast technology, applied in the biological field, can solve the problem of consuming manpower and material resources

Active Publication Date: 2020-12-29
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But the breeding of Zizania has been using a very old method, which consumes huge manpower an...

Method used

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  • Crispr/cas9 plasmid and its construction method and usage method
  • Crispr/cas9 plasmid and its construction method and usage method
  • Crispr/cas9 plasmid and its construction method and usage method

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0024] Example 1: Extraction of pMS7 plasmid

[0025] Use the Meiji Bio-endotoxin-free plasmid mini-extraction kit to extract the plasmid, and the specific steps are as follows:

[0026] 1. Inoculate the bacteria containing the pMS7 plasmid into a culture flask containing 15ml-20ml LB / carboxybenzyl culture solution, and culture on a shaker at 37°C for 12-16h to amplify the plasmid.

[0027] 2. Centrifuge at 3,000-5,000 x g for 10 minutes to collect 10-15ml of bacteria.

[0028] 3. Discard the medium, and gently tap on absorbent paper to absorb the residual liquid. Add 600µl Buffer E1 / RNase A, and vortex at high speed to fully resuspend the bacteria. RNase A must be added to Buffer E1 before use. Thorough resuspension of bacteria is critical to yield and no bacterial clumps should be visible after resuspension.

[0029] 4. Add 600µl Buffer E2 to the resuspension solution, invert and mix 6-8 times. Leave at room temperature for 1 to 2 minutes, and mix by inverting 2 to 3 ti...

example 2

[0040] Example 2: Linearization of the pMS7 plasmid

[0041] Enzyme digestion system (30 ul)

[0042] PMS7 plasmid DNA 4ul

[0043] Acc65I HF (neb) 1 ul

[0044] 1x Qcut Buffer 3 ul

[0045] dd H 2 O 22 ul

[0046] At 37°C, digest for 2h.

[0047] PCR products were detected by 1% agarose gel electrophoresis. After cutting the gel, it was recovered with the Magen Gel DNA Micro Recovery Kit. Store in a -20°C refrigerator for later use. pMS7 plasmid map as figure 1 shown. The pMS7 plasmid was digested with a single enzyme, such as image 3 As shown, the pMS7 plasmid was digested with the restriction endonuclease Acc65I to obtain a linearized fragment, which was recovered by rubber tapping to obtain the target fragment. The nucleotide sequence of the pMS7 plasmid is shown in SEQ ID No.1.

example 3

[0048] Example 3: Itd1 The long fragment of the gene sequence is ligated with the pMS7 vector cut by single enzyme

[0049] To complete this step, use the ClonExpress II OneStep Cloning Kit recombinant cloning kit produced by Nanjing Novizan Biotechnology Co., Ltd. Will Itd1 Partial fragment sequence of the gene (as shown in SEQ ID No.2), scaffold, and U6 terminator to synthesize a long fragment of Itd1-CRISPR, the nucleotide sequence of the Itd1-CRISPR is shown in SEQ ID No.4. The specific steps are as follows:

[0050] 1) Prepare the following reaction system on ice

[0051] Components (20ul)

[0052] pMS7 linearized vector 1.5ul

[0053] Itd1-CRISPR 1ul

[0054] 5×CEⅡ buffer 4 ul

[0055] Exnase II 2ul

[0056] dd H 2 O 11.5ul

[0057] (Note: The optimal amount of cloning vector used in the ClonExpress® II recombination reaction system is 0.03 pmol, and the optimal amount of insert used is 0.06 pmol (the molar ratio of vector to insert is 1:2). The DNA quality c...

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Abstract

The invention discloses CRISPR/Cas9 plasmid as well as a construction method and a use method of the CRISPR/Cas9 plasmid and belongs to the technical field of biotechnology. A nucleotide sequence of the CRISPR/Cas9 is shown in SEQ ID No.3. The CRISPR/Cas9 plasmid for artificial breeding of zizania latifolia is constructed with a CRISPR/Cas9 technology and converts ustilago esculenta, a gene knockout strain can be obtained, and the CRISPR/Cas9 plasmid can be more conveniently and efficiently applied to artificial breeding of the zizania latifolia.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a CRISPR / Cas9 plasmid, a construction method and a use method thereof. Background technique [0002] Ustilago smut ( Ustilago esculenta ) is a unique pathogenic fungus that infects Zizania plants ( Zizania latifolia ) After that, it can inhibit the heading and flowering of the plants and stimulate the expansion of the base of the stem to form edible wild rice stems. In East Asia and Southeast Asia, especially China, Zizania is a highly nutritious aquatic vegetable and an important medicinal material. However, the breeding of Zizania has been using very old methods, which cost a lot of manpower and material resources. Researchers have been working on optimizing the artificial breeding of Zizania. [0003] CRISPR / Cas9 technology is a gene editing technology that has developed rapidly in recent years, and has been successfully applied to genome editing in many plants. ...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N15/90C12N1/15A01G7/06
CPCA01G7/06C07K14/37C12N15/80C12N15/902
Inventor 张雅芬叶子弘于金梦夏文强俞晓平崔海峰
Owner CHINA JILIANG UNIV
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