Normalized cDNA library of ocean medaka specific tissue and preparation method thereof

A medaka and specific technology, applied in the field of cDNA library, can solve the problems of incomplete genetic information, high proportion of copies, no product seen, etc., and achieve the effect of convenient analysis and research

Inactive Publication Date: 2010-10-06
香港城市大学深圳研究院
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the genetic information of marine medaka fish is still incomplete. Searching professional cDNA library construction companies at home and abroad has not found related products; searching the genetic database of the National Library of the United States, the available genetic information is less than 30
[0003] The construction of cDNA library is an important technology in the field of molecular biology, but the biggest problem faced by cDNA library constructed by conventional methods is that the proportion of duplicate copies is too high in the process of high-throughput analysis. Gene expression levels vary widely in eukaryotic cells

Method used

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  • Normalized cDNA library of ocean medaka specific tissue and preparation method thereof
  • Normalized cDNA library of ocean medaka specific tissue and preparation method thereof
  • Normalized cDNA library of ocean medaka specific tissue and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Construction of a uniform cDNA library of liver tissue of marine medaka

[0043] The steps of this embodiment are as follows:

[0044] 1. Culture of Marine Medaka

[0045] The marine medaka fish comes from commercial farms in Taiwan, purchased and donated by the Department of Biology and Chemistry, City University of Hong Kong. The laboratory culture conditions of marine medaka fish are seawater dissolved oxygen level 5.0-7.0mg / L, water temperature 28±2℃, density setting 0.3-0.5L per fish, salt concentration 30mg / L, light cycle 14h lighting / 10h dark room, feeding 5 times a day, sex ratio male: female=1:1.5. Sexually mature adult fish with a body length of 3.5-4cm were selected as research objects in the experiment.

[0046] 2. Isolation of Marine Medaka Liver Tissue

[0047] Select 10 adult marine medaka fish, including 5 females and 5 males, kill them quickly by decapitating them, wash them twice with PBS buffer, separate the liver tissue aseptically, an...

Embodiment 2

[0058] Example 2 Construction of a homogeneous cDNA library of gonad tissue of marine medaka fish

[0059] Select 10 adult marine medaka fish, including 5 females and 5 males, kill them quickly by decapitating them, wash them twice with PBS buffer, separate the gonad tissue by aseptic operation, and place them in a bottle containing buffer. Wash twice, transfer to Eppendorf tubes, and collect mixed gonad tissues by centrifugation at 2000rpm / min at low speed. Other steps are the same as in Embodiment 1.

[0060] attached Figure 4 It is the result of 1% agarose gel electrophoresis of the total RNA of the gonad tissue of marine medaka fish. The 28SrRNA and 18S rRNA bands are bright and clear, and the 5S rRNA band is weak, indicating that the integrity of the total RNA is better.

[0061] The results of 1% agarose electrophoresis before and after normalization of LD-PCR amplification products of marine medaka gonad tissue cDNA library are shown in the appendix Figure 5 And at...

Embodiment 3

[0063] Example 3 Identification of the homogenized cDNA library of liver tissue of marine medaka fish

[0064] Randomly select 22 clones from the LB culture plate, inoculate them in 10ml of LB culture medium, cultivate them at 37°C for 28 hours, collect the cultures, and use the sequence primers of SEQ ID No.4 and SEQ ID No.5 to amplify the insert fragments. The increasing conditions are 95°C, 30 seconds; 65°C, 30 seconds; 72°C, 3 minutes; 30 cycles. 5ul of the product was subjected to 1% agarose gel electrophoresis, and the size of the inserted fragment was observed. The results are shown in the appendix Figure 7 , the 22 clones selected at random all have inserts, the size of which inserts ranges from 500bp-3kb, most of which are around 1kb-2kb. Sequencing at the same time, the sequencing primer sequences are SEQ ID No.4 and SEQ ID No.5, the sequencing results were compared in the gene bank, and one of the sequencing results was 99% consistent with the gene sequence gb|EF39...

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Abstract

The invention discloses a normalized cDNA library of ocean medaka specific tissues and a preparation method thereof, belonging to the field of cDNA libraries. The library is prepared from one or more specific tissues of ocean medaka. The preparation method of the normalized cDNA library comprises the following steps: extracting total RNA of the specific tissues of the ocean medaka, synthesizing cDNA polynucleotide products, re-hybridizing after cDNA denaturation, selectively degrading double-stranded components, amplifying normalized cDNA components through PCR and inserting amplified products in carriers. The normalized cDNA library of ocean medaka specific tissues lays a foundation for greatly finding and researching the expressing genes of the ocean medaka, and is of a great significance to the deep research on the expressing markers of the ocean medaka specific tissues.

Description

technical field [0001] The invention relates to the field of cDNA libraries, in particular to a uniform cDNA library of marine medaka fish-specific tissues and a preparation method thereof. Background technique [0002] Marine medaka (Oryzias Melastigma) is commonly found in the waters of China, North Korea, Japan, and India. Adults are about 3.5-4 cm long and have a life cycle of about 2-3 months. The genome is small, only half of the zebrafish genome , about 800Mb, mild in character, high in spawning rate, easy to observe, low in seawater consumption, more tolerant to seawater environmental pressure, and the liver and reproductive tissues are more sensitive to environmental pressure changes caused by hypoxia, so it is very suitable as a Model organisms are used in scientific research, especially for experimental research in marine environmental quality assessment and marine ecotoxicology. However, the genetic information of marine medaka fish is still incomplete. Searchin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B40/02C40B50/06C12N15/70C12R1/19
Inventor 杨梦甦陈瑶方志俊张琦曾志雄胡绍燊
Owner 香港城市大学深圳研究院
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