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Cotton leaf DNA extraction method

A leaf and cotton technology, applied in the field of high-throughput, rapid and low-cost extraction of cotton leaf DNA, can solve the problems of cumbersome cotton leaf DNA extraction process, high cost of reagents and consumables, poor effect, etc., and achieves saving of labeled sampling tubes. The effect of less time, less sample tissue, and convenient operation

Inactive Publication Date: 2015-02-25
INST OF ECONOMIC CROP HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problems of cumbersome cotton leaf DNA extraction process, long time-consuming, high cost of reagent consumables and poor effect of various TPS methods on cotton leaf DNA extraction in the traditional method, and provides a cotton leaf DNA extraction method. DNA extraction method, which is fast, simple, and low-cost, and can extract DNA from cotton leaves in different periods in large quantities

Method used

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  • Cotton leaf DNA extraction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] A method for extracting cotton leaf DNA, comprising the following steps:

[0046] Cotton cotyledon DNA extraction

[0047] After the individual plants to be tested are listed and numbered, a 96-well PCR plate is used for sampling. 1 / 4-1 / 2 square centimeter cotyledons were put into 96-well PCR plate in sequence, covered tightly with a silicone sealing cushion, and stored in an ice box.

[0048] Add 70 μl of TPS extraction buffer to the wells of the PCR plate, grind the cotyledons with a micro electric drill until they are fully broken, cover with a silicone sealing cushion, and bathe in water at 95°C for 20 minutes, then centrifuge the PCR plate containing the samples at a speed of 3000 rpm 2 minute. Take 20 μl of supernatant, add 180 μl of sterilized TE buffer, pipette up and down with a pipette tip to mix the DNA solution, and obtain a DNA sample that can be used directly.

[0049] The formula of the TPS extraction buffer is: 0.1M Tris-HCL, 0.01M EDTA, 1.2M / L KCL, 2...

Embodiment 2

[0056] A method for extracting cotton leaf DNA, comprising the following steps:

[0057] (1) DNA extraction after cotyledon pretreatment

[0058] After the individual plants to be tested are listed and numbered, a 96-well PCR plate is used for sampling. Take cotyledons of 1 / 4-1 / 2 square centimeters, put them in a 96-well PCR plate in sequence, cover them tightly with a silicone sealing cushion, and store them in an ice box.

[0059] Add 70 μl of pretreatment buffer to the wells of the PCR plate, and grind the cotyledons with a micro hand drill until they are fully broken. Cover with a silicone sealing cushion, and centrifuge the PCR plate containing the samples at a speed of 3000 rpm for 2 minutes. Take out the PCR plate, peel off the sealing pad, cover the PCR plate with multiple layers of absorbent paper, invert the PCR plate, and let the absorbent paper absorb the supernatant. Add 70 μl of TPS extraction buffer to the wells of the PCR plate, cover with a silica gel seali...

Embodiment 3

[0068] A method for extracting cotton leaf DNA, comprising the following steps:

[0069] (1) DNA extraction after pretreatment of adult leaves

[0070] After the individual plants to be tested in the field were listed and numbered, a 96-well PCR plate was used for field sampling. Take the leaves of 2-3 leaves and 1 / 4 square centimeters of the cotton plants before topping, put them into 96-well PCR plates in sequence, cover them tightly with silica gel sealing cushions after taking them, and store them in an ice box.

[0071] Add 70 μl of pretreatment buffer to the wells of the PCR plate, grind the leaves with a micro electric drill until they are fully broken, cover with a silicone sealing cushion, and centrifuge the PCR plate containing the samples at a speed of 3000 rpm for 2 minutes. Take out the PCR plate, peel off the sealing pad, cover the PCR plate with multiple layers of absorbent paper, invert the PCR plate, and let the absorbent paper absorb the supernatant. Add 70...

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Abstract

The invention provides a cotton leaf DNA extraction method. The cotton leaf DNA extraction method includes the steps that pretreated buffer solution is added into cotton cotyledons or inverted adult plant leaves; after grinding, supernatant is centrifugally removed; buffer solution is extracted by adding TPS (toughened polystyrene) under water and metal bath of the temperature of 90 to 97 degrees Celsius; after centrifugation, supernatant with leaf DNA (deoxyribonucleic acid) is obtained. Cotton leaves from any periods after emergence and before topping can be selected as DNA extraction material. Compared with other fast DNA extraction methods, the cotton leaf DNA extraction method is good in DNA quality and suitable for PCR (polymerase chain reaction) analysis; compared with an existing cotton leaf DNA extraction method of CTAB (cetyl trimethyl ammonium bromide), steps are simplified, sampling and DNA extraction time are saved by 80 to 90 percent, costs are reduced by 90 percent. The cotton leaf DNA extraction method is applicable to large and fast extraction of low-cost DNA samples during marker-assisted molecular breeding process.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a high-throughput, fast and low-cost method for extracting DNA from cotton leaves. Background technique [0002] In recent years, with the in-depth research on the location of crop quantitative trait loci, molecular marker technology has been more and more widely used in crop marker-assisted breeding by scientific research and breeding units. In crop marker-assisted breeding programs, high-throughput, rapid, and low-cost extraction of DNA from individual crop plants has always been an ideal goal for scientific researchers and breeders. In the work of molecular marker-assisted selection in cotton, DNA samples are generally extracted from the leaves of cotton plants. Different from the DNA of other plants, cotton leaves are not only rich in protein, but also contain secondary metabolites such as gossypol, polysaccharides, and tannins. When an irreversible reaction occurs, a brown col...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 秦鸿德易先达张成王小娇焦春海陈敏
Owner INST OF ECONOMIC CROP HUBEI ACADEMY OF AGRI SCI