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Construction method and application of two-color fluorescence report carrier

A two-color fluorescence and carrier technology, applied in the fields of molecular biology and antiviral research, can solve the problems of inaccuracy, affecting the accuracy of quantification, and the inability to accurately project the inhibition efficiency of siRNA

Inactive Publication Date: 2008-09-10
WUHAN UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, both of these two methods have a major disadvantage: they cannot accurately reflect the inhibitory efficiency of siRNA when quantifying.
Therefore, in the above two types of methods, it is obviously inaccurate to use the entire cell population instead of selecting cells that meet the requirements for quantitative analysis.
Moreover, in the first method, the cells are not tested under physiological conditions, so the test cells must be lysed, which undoubtedly affects the accuracy of quantification

Method used

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  • Construction method and application of two-color fluorescence report carrier
  • Construction method and application of two-color fluorescence report carrier
  • Construction method and application of two-color fluorescence report carrier

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Experimental program
Comparison scheme
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Embodiment 1

[0096] Embodiment 1PCR amplification has the ampicillin resistance gene sequence of promoter and transcription termination signal

[0097] Synthesis of amplification primers: primers were designed according to the ORF of the ampicillin resistance gene and the vector, and detected by primerpremier 5.0 software (product of Premier Company in Canada). The 5'primer P1 used is: CCTAGAT CTTAAG AAATTAAAAATGAAGTTT (the underline is the introduced Bgl II restriction site); 3'primer P2 is: TAATAATGGTTT CACGTAGTG CAGGTGGC (underlined is the introduced Dra III restriction site). The primers used in the present invention were all synthesized by Wuhan Saibaisheng Company. Using pUC18 (product of Takara Company) as a template, using upstream primers and downstream primers, a DNA sequence from 1566 to 2617 in pUC18 was amplified using a PCR machine.

[0098] PCR system components: 5μl 10×1 # buffer, 5 μl dNTP (2mM), 1 μl primer P1 (10 μM), 1 μl primer P2 (10 μM), 0.1 μl template pUC18, ...

Embodiment 2

[0101] Example 2 Recovery and purification of ampicillin resistance gene PCR product with promoter and transcription termination signal

[0102] (1) Carry out electrophoresis (1×TAE) with 1.0% agarose gel to the PCR product among the embodiment 1, observe electrophoresis situation with ultraviolet light, when the DNA band to be recovered is completely separated from other bands, stop electrophoresis, in Cut off the band to be recovered with a razor blade under ultraviolet light, and purify it with a PCR product purification kit. (2) Crush the glue in an Eppendorf tube, add an equal volume of sol solution Binding Buffer, bathe in water at 65°C for 7 minutes, and shake the Eppendorf tube every 2 minutes until the glue is completely melted. (3) Add the melted sample to the chromatographic column, centrifuge at 12000 rpm for 1 min, and discard the liquid. (4) Add 300 μl BindingBuffer, centrifuge and discard the liquid. (5) Add 750μl Washing Buffer, centrifuge and discard the liq...

Embodiment 3

[0103] Example 3 Plasmid pEGFP-N1 is single-digested by Afl II

[0104] Reaction system: 5 μl 10×0 buffer; pEGFP-N1 plasma 15 μl; 5 μl Afl II enzyme; add sterile water ddH 2 0 to 50 μl.

[0105] Reaction process: Mix and mix the components of the reaction system on ice, centrifuge briefly at 5000rpm for 10s, place in a water bath at 37°C for 2-4h, and use 1% agarose gel electrophoresis to cut out the target band under UV light , Recovery and purification were carried out with the Gel Recovery Kit. Finally, it was eluted with 30 μl sterile water. Take 2 μl samples for 1% agarose electrophoresis, and store the rest at -20°C for later use. In this step, pEGFP-N1 was cut by Afl II single enzyme.

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Abstract

The invention discloses a method for constructing a dual-color fluorescence reporter vector and application thereof. The sequence of the dual-color fluorescence reporter vector is a nucleotide sequence as shown in SEQ ID NO.1. The method for constructing the dual-color fluorescence reporter vector comprises the following steps of: A. the construction of derived plasmid pEGFP-N1-Ampicillin; B. the construction of derived plasmid pMD18-EGFP; C. the construction of derived plasmid pMD18-EGFP-polyA; D. the construction of derived plasmid pEGFP / EGFP; E. the final construction of target plasmid pEGFP / DsRed; F. the preparation of a derived dual-color fluorescence vector pEGFP / DsRed-HBx of the dual-color fluorescence reporter vector pEGFP / DsRed. The method has the advantages of easy implementation and convenient operation, and the vector can be applied to high throughput screening and quantitative siRNA efficiency.

Description

technical field [0001] The invention relates to the fields of molecular biology and antiviral research, and can screen out antiviral siRNA drugs with high throughput. More specifically, the present invention relates to a two-color fluorescent reporter carrier, a construction method of the two-color fluorescent reporter carrier, and the application of the invention in high-throughput and accurate evaluation of siRNA efficiency. Background technique [0002] RNA interference (RNAi) is a molecular mechanism that is conserved from yeast to humans: one strand of double-stranded small interfering RNA (siRNA) is integrated into RISC (RNA-Induced Silencing Complex), when this strand finds When paired with the target mRNA, the target mRNA will be cleaved and then degraded by RISC sequence specificity (Fire A, Xu SQ, Montgomery MK, Kostas SA, Driver SE, Mello CC 1998 Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806-811.). Now ...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/64C12Q1/68G01N21/64
Inventor 张翼童文平王欣欣谢茂华
Owner WUHAN UNIV
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