Method for quickly constructing siRNA carrier by using pSilencer plasmid

A carrier and fast technology, applied in the direction of using carrier to introduce foreign genetic material, recombinant DNA technology, etc., can solve the problems of long fragments and high cost of siRNA, and achieve the effect of reducing cost, reducing the probability of error, and saving construction time

Inactive Publication Date: 2009-11-18
SHANDONG UNIV
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  • Description
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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 bc1212 gene, find the target of the interfering gene (GCTGGGCATCAGTGAGGAC), and synthesize the upper and lower gene sequences 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 gene sequence: 5ug ...

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Abstract

The invention discloses a method for rapidly constructing an siRNA carrier by using pSilencer plasmid. The steps include the construction of subcloning vector, the synthesis method of siRNA used in the rapid screening process, the rapid screening method of positive clones containing siRNA, and the like. Compared with conventional vector construction methods, this method is more effective, faster, and more economical to obtain the required small-scale gene vectors, and is especially suitable for functional verification analysis of large-scale gene screening results such as gene chips, SSH, and SAGE. , and research on the search for effective inhibitory sequence siRNA and other RNA interference mechanisms.

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 Patents(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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