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Short-term preservation method of rubber tree leaves for RNA (ribonucleic acid) extraction

A preservation method and technology of tree leaves, applied in the biological field, can solve the problems of easy volatilization, high price, cumbersome operation, etc., and achieve the effects of less irritating smell, preventing oxidation, and avoiding browning

Inactive Publication Date: 2012-12-05
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, carrying liquid nitrogen or dry ice sampling in the field has disadvantages such as expensive, bulky tank, cumbersome operation, volatile, high risk, etc., and has failed to achieve simple, economical, safe, and efficient effects.

Method used

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  • Short-term preservation method of rubber tree leaves for RNA (ribonucleic acid) extraction
  • Short-term preservation method of rubber tree leaves for RNA (ribonucleic acid) extraction
  • Short-term preservation method of rubber tree leaves for RNA (ribonucleic acid) extraction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 1. After short-term or long-term storage, take out 1g of fresh leaves from the storage buffer and refrigerator (-70℃~-80℃) respectively, put the leaves into a pre-cooled mortar, and grind them into fine pieces in liquid nitrogen. Powder, transferred to two 5ml centrifuge tubes. (about 0.5g requires a 5ml centrifuge tube)

[0028] 2. Add 2.5ml of extraction buffer (200mM Sodium Borate PH9.0, 30mM EGTA, 1% SDS, 10mM DTT, 1% DOC, 2% PVP-40) preheated to 80°C into each 5ml tube, homogenate 2 min. (Use DEPC-H for homogenizing rotor first) 2 O wash several times, at least 3 times)

[0029] 3. Transfer the homogenate to a new centrifuge tube added with 200μl 20mg / ml proteinase K, and incubate at 100rpm in a shaker at 42°C for 1.5h.

[0030] 4. Add 500μl 1.6mol / L KCl to a final concentration of 160mmol / L, and keep in ice bath for 1h.

[0031] 5. Centrifuge at 4°C, 12000rpm for 20min, (or centrifuge at 4°C, 10000rpm for 30min), transfer the supernatant to another new tube, ...

Embodiment 2

[0041] 1. Take the samples collected in the field or sent from other places out of the foam box, or take out the samples from the -70~-80°C refrigerator, select 800mg of fresh young tissue of the rubber tree in a mortar, add liquid nitrogen to fully grind into a powder and transferred to a 10ml centrifuge tube.

[0042] 2. Add 4ml of RNA extraction solution (1.4mol / L NaCl, 0.1mol / L Tris·HCl (after pH8.0 sterilization), 20mmol / L EDTA, 2% CTAB, 2% PVP, 1 %(V / V) β-Me (add when used), the above-mentioned medicines except Tris HCl first use 0.1% DEPC-H 2 O preparation, shake for 12hs, and then sterilize, then add 0.1mol / L Tris, adjust the pH value to 8.0 with HCl, 320μl β-Me, shake and mix, 65℃ water bath for about 30min, mix 2-3 times in the middle.

[0043] 3. Add 0.6 times the volume of chloroform, mix well back and forth, and then ice bath for 10 minutes.

[0044] 4. Centrifuge at 4°C and 9000rpm for 20min, and transfer the supernatant to another new 10ml centrifuge tube.

...

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Abstract

The invention relates to a short-term preservation method of rubber tree leaves for RNA (ribonucleic acid) extraction. The short-term preservation method of rubber tree leaves for RNA extraction includes: collecting tender leaves of rubber tree in light bronze-colored stage, deep bronze-colored stage, discoloring stage or light green stage, loading the leaves in a centrifugal tube, adding storagebuffer, placing the centrifugal tube in an ice box or ice barrel containing ice cubes at 0-4 DEG C, bringing the box or barrel to a lab, and placing the box or barrel on an ice rock at 0-4 DEG C for standby. Tissues of rubber tree leaves are stored by the storage buffer and low-temperature storage, so that freshness of the rubber tree leaves can be maintained in a short time effectively and remote sampling and material transport and storage are facilitated. The short-term preservation method has the advantages of simple process, low cost, effectiveness and the like, and quality security in sample RNA extraction is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a leaf tissue preservation method, in particular to a short-term preservation method for rubber tree leaves used for RNA extraction. Background technique [0002] Rubber tree (Hevea spp.) is a perennial cross-pollinated tree of Euphorbiaceae (Euphorbiaceae). It is a special forest tree that produces rubber in tropical areas and is an important economic crop. my country's large-scale natural rubber bases are mainly distributed in the three provinces of Hainan, Yunnan and Guangdong. Since the 1990s, with the rapid development of molecular biology, research on rubber trees has gradually entered the molecular level. Research on the expression and regulation of genes related to rubber tree yield, stress resistance, growth and metabolism, etc. from the transcriptional level is an important tool for rubber tree expression quantitative trait locus (eQTL) mapping, transgenic breeding, identifi...

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王祥军
Owner RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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