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Method for quickly constructing siRNA carrier by using pSilencer plasmid

A vector and rapid technology, applied in the introduction of foreign genetic material using vectors, recombinant DNA technology, etc., can solve the problems of long fragments and high cost of siRNA, and achieve the effect of reducing costs, reducing the probability of errors, and saving construction time.

Inactive Publication Date: 2008-01-30
SHANDONG UNIV
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AI Technical Summary

Problems solved by technology

The cost of siRNA synthesized by the above method is higher due to the longer synthesized fragments

Method used

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  • Method for quickly constructing siRNA carrier by using pSilencer plasmid
  • Method for quickly constructing siRNA carrier by using pSilencer plasmid
  • Method for quickly constructing siRNA carrier by using pSilencer plasmid

Examples

Experimental program
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Effect test

Embodiment 1

[0041] 1. Synthesis of siRNA gene fragments and PCR completion reaction:

[0042] 1) According to the sequence of the bcl2l2 gene, the target of the interfering gene (GCTGGGCATCAGTGAGGAC) was found, and the upstream and downstream gene sequences were synthesized according to the instructions of pSilencer plasmid construction. Including adding a circular base sequence in the middle of the sequence, adding HindIII and BamHI enzyme cutting sites upstream and downstream, and adding protective bases at both ends of the enzyme cutting site to facilitate subsequent enzyme cutting (by Shanghai Boya company synthesis).

[0043] Upstream gene sequence (53bp):

[0044] 5'-GCGGATCCGCTGGGCATCAGTGAGGACATTCAAGAGATGTCCTCACTGATGCC-3'

[0045] Downstream gene sequence (53bp):

[0046] 5'-GCAAGCTTTTCCAAAAACTGGGCATCAGTGAGGACATCTCTTGAATGTCCTC-3'

[0047]2) PCR reaction to complete the siRNA gene fragment:

[0048] Pyrobest DNA Polymerase (purchased from Takara): 2.5U / 0.5ul

[0049] Upstream ...

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Abstract

The invention discloses a method by using pSilencer plasmid to quickly establish a siRNA carrier. The invention comprises the steps of: the establishment of a sub-cloning carrier, a siRNA synthesis method used for quick screening course and quick screening methods of positive clone with siRNA, etc. Compared with traditional carrier establishing method, the method of the invention can more effectively, quickly and economically obtain required small reference gene carrier and can be more particularly applicable for function verification and analysis for large-scaled primary screening results of genes such as gene chip, SSH and SAGE, etc. and research of RNA reference mechanism for seeking effective inhibiting sequence siRNA, etc.

Description

technical field [0001] The invention relates to a method for rapidly constructing siRNA vectors, in particular to a method for rapidly constructing siRNA vectors using pSilencer plasmids in a pSilencer vector system. technical background [0002] RNA interference technology is a powerful tool for studying gene function, and "Science" magazine called RNAi "a major breakthrough in 2002" (Couzin, 2002). Andrew Fire and Craig Mello won the 2006 Nobel Prize in Medicine for their discovery. The preparation technology of small interfering RNA mainly includes: chemical synthesis method, construction of siRNA expression vector, in vitro transcription method, preparation of siRNA mixture, siRNA expression cassette, wherein the siRNA generated by the vector construction method can be continuously expressed in cells for several weeks or even longer. Time, but also easily accepted by molecular biology experimenters. The pSilencer series of plasmids produced by Ambion are commonly used ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63
Inventor 孙汶生张之勇韩丽辉曹莉莉鞠瑛杜娟马恬侯楠曲忠花刘玉刚崔敏梁晓红
Owner SHANDONG UNIV
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