Quick batch preparation method of cotton genome DNA (deoxyribonucleic acid) suitable for PCR (polymerase chain reaction)

A cotton genome, fast technology, applied in the direction of DNA preparation, recombinant DNA technology, etc., can solve the problems of complex extraction steps, time-consuming and labor-consuming, and achieve the effect of reducing the types of use, less materials, and simple steps

Inactive Publication Date: 2013-02-13
XINJIANG INST OF ECOLOGY & GEOGRAPHY CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The invention solves the problem of complicated, time-consuming and labor-intensive extraction steps of cotton genome DNA, and provides a method for rapidly and batch-extracting DNA that meets the requirements of PCR detection. The method can efficiently, quickly and simply extract DNA from cotton leaves The extraction meets the template DNA of PCR detection, which can be applied to molecular biology detection in the process of genetically modified cotton breeding and safety declaration.

Method used

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  • Quick batch preparation method of cotton genome DNA (deoxyribonucleic acid) suitable for PCR (polymerase chain reaction)
  • Quick batch preparation method of cotton genome DNA (deoxyribonucleic acid) suitable for PCR (polymerase chain reaction)
  • Quick batch preparation method of cotton genome DNA (deoxyribonucleic acid) suitable for PCR (polymerase chain reaction)

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Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1 (method of the present invention:)

[0032] Collection of samples: Cotton leaves are young leaves taken from the top of field cotton plants, put into 2mL centrifuge tubes, put them into a liquid nitrogen tank, and bring them back to the laboratory for storage in a -80°C refrigerator, or freshly collected materials from any indoor cultivated cotton; 0.05-0.15g young leaves of cotton lines with transgenic ScALDH gene (1450bp), EsDREB gene (750bp), TcLEA gene (310bp) and control lines planted in the laboratory and tested positive by kanamycin were put into 2mL centrifuge tube (produced by Sangon Bioengineering (Shanghai) Co., Ltd.), immediately put it into a liquid nitrogen tank and bring it back to the laboratory for storage at -80°C refrigerator;

[0033] Extraction of Genomic DNA from Transgenic Cotton:

[0034] Take the healthy stored cotton leaves and put them into a 2mL centrifuge tube, add 600μL of the extract solution consisting of NaCl 500mmol / L, tri...

Embodiment 2

[0045] Embodiment 2 (contrast with the present invention)

[0046] The sample used in this embodiment is the same as in Example 1 with collection method;

[0047] Comparison method 1:

[0048] Extraction of Genomic DNA from Transgenic Cotton:

[0049] Take a 2mL centrifuge tube with cotton leaves, add 600μL extract solution to the centrifuge tube: NaCl500mmol / L, tris-hydroxymethylaminomethane (Tris-Cl)50mmol / L, pH=8.0, sodium dodecyl sulfate (SDS) 2%, polyvinylpolypyrrolidone (PVP) 6%, β-mercaptoethanol 1%, and RNase A with a final concentration of 50 μg / mL, and add two steel balls (included with the cell disruptor), with a particle size of 4-6mm;

[0050] Put the centrifuge tube into a mixing grinder and crush it at a frequency of 30HZ for 5min to achieve crushing of the leaves. Put the treated centrifuge tube into a water bath at a temperature of 65°C for 10min, centrifuge at 7000rpm for 2min, and transfer 400μL of the supernatant to Store in a new centrifuge tube at -20°...

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Abstract

The invention discloses a quick batch preparation method of cotton genome DNA (deoxyribonucleic acid) suitable for PCR (polymerase chain reaction), relating to the molecular detection of genetically modified crops in the field of crop breeding. The method comprises the following steps of: putting the tender leaves at the top of a cotton plant into a centrifuge tube; adding an extracting solution and steel balls; breaking the sample; performing treatment in a water bath; adding a leaching solution for leaching; precipitating DNA with isopropyl alcohol; washing with ethanol; and dissolving the precipitate in water to obtain the extracted DNA. The method disclosed by the invention solves the problems of complicated steps and time and labor consumption in cotton genome DNA extraction; the invention provides a method capable of quickly extracting the DNA meeting the requirements of PCR detection in batches; and the method can be applied to the molecular biological detection in the links of genetically modified cotton breeding, safety report and the like.

Description

technical field [0001] The invention relates to a rapid and batch preparation method of cotton genome DNA for PCR. Background technique [0002] Cotton is an important economic crop in my country and even in the world. At present, my country is the largest cotton producer in the world. In recent years, with the continuous development of molecular biology technology and genetic breeding technology, a large number of genes with resistance to biotic stress (insect resistance) and abiotic stress (herbicide resistance, drought resistance) have been successfully transferred into cotton. These high-quality genetic resources have made significant contributions to high crop yields and reduced environmental pollution. However, when cultivating new varieties, the field testing of each generation of transgenic cotton has always been a major problem for breeders and molecular biology researchers. Because the workload of this link is heavy, time-consuming and labor-intensive. It is oft...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 张道远李小双李海燕
Owner XINJIANG INST OF ECOLOGY & GEOGRAPHY CHINESE ACAD OF SCI
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