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Method for preserving rubber tree leaf tissues extracted by DNA (deoxyribonucleic acid)

A preservation method and technology of tree leaves, applied in the biological field, can solve the problems of inconvenient carrying, high power consumption, high heat dissipation, etc., and achieve the effect of facilitating remote sampling, avoiding browning, and simple process

Inactive Publication Date: 2011-04-13
RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ultra-low temperature refrigerators are inconvenient to carry and high in cost due to their disadvantages such as high price, high net weight, high noise, high power consumption, high heat dissipation, and the working room temperature requirement of 16-32°C, and fail to achieve the effect of saving daily expenses.

Method used

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  • Method for preserving rubber tree leaf tissues extracted by DNA (deoxyribonucleic acid)
  • Method for preserving rubber tree leaf tissues extracted by DNA (deoxyribonucleic acid)
  • Method for preserving rubber tree leaf tissues extracted by DNA (deoxyribonucleic acid)

Examples

Experimental program
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Effect test

Embodiment 1

[0024] 1. Take out the sample after short-term or long-term storage, melt it at room temperature, transfer a part of the liquid sample to a 1.5ml centrifuge tube with a 1ml pipette tip with the front part cut off, and centrifuge at 12000rpm for 15min at 4°C.

[0025] 2. Discard the supernatant, add 650ul 2×CTAB extraction buffer preheated at 65°C to the precipitation (2×CTAB extraction buffer composition: 0.1M Tris-HCl, 20mM Na-EDTA, 1.4M NaCl, 2% CTAB, 2% PVP, 1% (V / V) B-Me), vortex or use a clean toothpick to stir the precipitate, and incubate at 65°C for 30-60 minutes (the time of incubating can be as long as several hours).

[0026] 3. Add an equal volume of chloroform: isoamyl alcohol (24:1), invert back and forth more than 50 times, let stand at room temperature for 10 minutes, centrifuge at 10,000 rpm for 15 minutes at 15°C or room temperature, and carefully transfer the supernatant to a new centrifuge tube (care should be taken to prevent excessive DNA fragmentation). ...

Embodiment 2

[0033] 1. Take the sample out of the mailed foam box or -20°C refrigerator. After melting at room temperature, transfer a part of the liquid sample to a 10ml centrifuge tube with the blue pipette tip with the front end cut off. Centrifuge at 10000rpm for 20min, discard the supernatant.

[0034] 2. Add 3ml of 65°C preheated 2% SDS extraction buffer (2% SDS extraction buffer composition: 100mM Tris-HCl, pH 8.0, 50mM EDTA, pH 8.0, 100mMNaCl, 2% SDS, 1% ( V / V) B-Me), and loosen it with a copper wire, vortex to mix, and take a water bath at 65°C for 30-45 minutes (the water bath time should not be too long, it will easily degrade if it is too long).

[0035] 3. Add 3ml of chloroform:isoamyl alcohol (24:1) and turn over more than 50 times, let stand at room temperature for 10min, centrifuge at 15°C and 10,000rpm for 20min, transfer the supernatant into a 10ml centrifuge tube (handle with care to prevent DNA too many breaks).

[0036] 4. Add 0.6V pre-cooled isopropanol, turn over s...

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PUM

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Abstract

The invention belongs to the field of biotechnologies and relates to a method for preserving rubber tree leaf tissues extracted by DNA (deoxyribonucleic acid). The method comprises the following steps: placing collected rubber tree leaves in a pre-cooled mortar, and utilizing liquid nitrogen for grinding until the leaves become powder; and then transferring the powdery leaf tissues into a centrifugal tube containing storage buffer solution, uniformly mixing and placing in a low-temperature environment for preservation. The method which combines the storage buffer solution with low-temperaturerefrigeration is utilized for preserving the rubber tree leaf tissues, thereby effectively realizing the freshness of the rubber tree leaves for a long time, facilitating long-distance sampling, material transportation and storage, and having the characteristics of simplicity in process, low cost, good effects and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for preserving leaf tissue, in particular to a method for preserving rubber tree leaf tissue for DNA extraction. Background technique [0002] Brasilian rubber tree (Hevea brailiensis), Euphorbiaceae (Euphorbiaceae) rubber tree genus (Hevea), is a rubber tree in tropical areas and is an important economic crop. Since the 1990s, with the rapid development of molecular biology, research on rubber trees has gradually entered the molecular level. Research on the genetic basis and genetic resources of rubber trees at the molecular level is an important means for the development and utilization of germplasm resources of the genus Hevea, species identification, and assisted breeding. Foundation. [0003] At present, the conventional methods for extracting genomic DNA from rubber tree leaves mainly include CTAB method and SDS method. The concentration and purity of DNA extracted by ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N3/00
Inventor 吴春太黄华孙李维国高新生张晓飞
Owner RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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