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Method for constructing eukaryote cDNA library and special primers thereof

A eukaryotic library technology, applied in the field of eukaryotic cDNA library construction, can solve the problems of information loss, affecting the accuracy of results, etc., and achieve the effect of reducing difficulty

Active Publication Date: 2011-02-16
北京宝盈同汇生物技术有限公司
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to construct a cDNA library for existing eukaryotic organisms, which requires multiple use of endonuclease and DNA ligase to carry out enzyme digestion or enzyme linkage reaction, and after the reaction, a series of complicated procedures such as recovery and purification are required. Operation, resulting in the loss of part of the information in the sample, affecting the accuracy of the results and other defects, providing a simple method and special primers for the construction of eukaryotic cDNA libraries without the use of endonucleases and DNA ligases

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  • Method for constructing eukaryote cDNA library and special primers thereof
  • Method for constructing eukaryote cDNA library and special primers thereof
  • Method for constructing eukaryote cDNA library and special primers thereof

Examples

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Embodiment 1

[0043] Example 1: (Construction of cDNA library in peripheral blood of Holstein cows after heat stress treatment)

[0044] 2 mL of Holstein cow tail vein blood was collected with vacuum blood collection tubes (EDTA anticoagulant tubes) (BD). After heat-shock incubation at 42°C for 4 hours, total RNA was extracted from peripheral blood by Trizol (Invitrogen) method. 0.8 μg of total RNA was used as a template for reverse transcription, and SuperScript II reverse transcriptase (Invitrogen) was used for the reverse transcription reaction, and 3RRT and 5RRT primers were used to carry out the reverse transcription reaction of the sample total RNA. After the reverse transcription, 1 μL of the reverse transcription product was used as a template for PCR amplification, and the double-stranded cDNA was amplified with Ex Hot Start DNA polymerase (TaKaRa) and primers 3AP and 5AP. Use 1% agarose gel electrophoresis to separate the double-stranded cDNA, and cut the double-stranded cDNA with...

Embodiment 2

[0072] Embodiment 2: (tobacco leaf cDNA library construction)

[0073]Tobacco leaves were collected, and the total RNA of tobacco leaves was extracted by Trizol (Invitrogen) method. 4.8 μg of total RNA was used as a template for reverse transcription, and SuperScript II reverse transcriptase (Invitrogen) was used for the reverse transcription reaction, and 3RRT and 5RRT primers were used to carry out the reverse transcription reaction of the sample total RNA. After the reverse transcription, 1 μL of the reverse transcription product was used as a template for PCR amplification, and the double-stranded cDNA was amplified with Ex Hot Start DNA polymerase (TaKaRa) and primers 3AP and 5AP. Use 1% agarose gel electrophoresis for electrophoretic separation of double-stranded cDNA, and cut double-stranded cDNA with a molecular weight of 500-5000bp in the agarose gel to remove small fragment products less than 500bp or incomplete synthesis; use Omega's E.Z.N. The gel recovery kit i...

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Abstract

The invention discloses a method for constructing a eukaryote cDNA library and special primers thereof, belonging to the technical field of genetic engineering. The special primers are arranged in two pairs in total, one pair comprises 3RRT and 5RRT in a sequence table, and the other pair comprises 3AP and 5AP in the sequence table. The method comprises the following steps: 1) taking total RNA asa template, and taking the 3RRT and the 5RRT in the sequence table as the primers for synthesizing cDNA of a first chain; 2) using the 3AP and the 5AP in the sequence table for synthesizing the cDNA of double chains; and 3) carrying out agarose gel electrophoresis to remove small-fragment products which are synthesized incompletely, recombining and connecting the cDNA of the double chains with a long-fragment PCR cloning vector of pCR-XL-TOPO, further transforming a connection product to host Escherichia coli XL-Gold competent cells and finally completing the construction of the cDNA library.The method and the special primers can be commonly used for constructing the cDNA libraries for eukaryote samples, wherein mRNAs of the eukaryote samples have the polyA tail ends.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a simple method capable of constructing a eukaryotic cDNA library without endonuclease and DNA ligase and special primers thereof. Background technique [0002] Since the first cDNA library came out in the 1970s, the construction of cDNA library has become an indispensable technical means in the research of molecular biology, functional genomics and isolation and cloning of excellent resource genes. Restricted by the technical level at that time, the early cDNA libraries generally had the defects of low connection efficiency, difficulty in amplification and preservation. In recent years, new methods for cDNA library construction have emerged in an endless stream, but the purpose is to simplify the operation steps of cDNA library construction and meet the needs of research. [0003] The basic principle of cDNA library construction is to use purified Poly(A) mRNA as a ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12N1/21C12N15/10C12N15/11
Inventor 宋雪梅蒋永清燕飞陈剑平姜俊芳
Owner 北京宝盈同汇生物技术有限公司
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