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Method for extracting DNA (deoxyribonucleic acid) from small amount of grain oil

A grain and oil, a small amount of technology, applied in DNA preparation, recombinant DNA technology, biochemical equipment and methods, etc., can solve problems such as easy DNA loss, difficult DNA for grain and oil, and difficulty in detection.

Inactive Publication Date: 2017-10-20
北京爱普拜生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this method is that the large-volume oil extraction process introduces a large volume of aqueous buffer solution, resulting in too large a volume of the aqueous phase after the oil phase is separated from the aqueous phase, forcing DNA precipitation to be carried out only in 50mL centrifuge tubes
In addition, small-scale laboratories or testing institutions generally do not have high-speed centrifuges suitable for 50mL centrifuge tubes, which also increases the difficulty of grain and oil DNA extraction
However, although the 50mL centrifuge tube system provides the convenience of the experiment, due to the large area at the bottom of the 50mL centrifuge tube, it is not easy to see the precipitate after centrifugation, and it is easy to lose DNA during elution. eluent, which in turn reduces the DNA concentration after elution, making subsequent detection extremely difficult
Therefore, it is difficult to obtain oil DNA that can be used for detection using this method

Method used

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  • Method for extracting DNA (deoxyribonucleic acid) from small amount of grain oil
  • Method for extracting DNA (deoxyribonucleic acid) from small amount of grain oil
  • Method for extracting DNA (deoxyribonucleic acid) from small amount of grain oil

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 uses 1.5mL extraction system to extract DNA from four-grade transgenic rapeseed oil:

[0049] experimental method

[0050]1. Take a 2.0mL EP tube, add 1mL four-grade rapeseed oil and 400μL TE buffer, invert and mix for 5min, centrifuge at 12000rpm at room temperature for 1min, remove the upper layer of grease; add 1mL four-grade rapeseed oil again, repeat the above process 10-20 times ;Remove the upper layer of grease and leave some, transfer the lower layer of TE buffer to a new EP tube, collect about 1mL of the aqueous phase, and the enrichment is complete. Use APEXBIO's oil DNA extraction kit to extract about 1mL of the above solution, mainly: add 400μL bufferAP1, mix well and incubate at 65℃ for 10min, mix well 2-3 times during the period; add 130μL buffer AP3, mix well After uniformity, place it on ice and incubate for 5 minutes, then centrifuge at 20,000g for 5 minutes, transfer all the supernatant to the impurity removal column, centrifuge at 14,000rpm...

Embodiment 2

[0070] Example 2 Using 1.5mL extraction system to extract DNA from transgenic soybean crude oil

[0071] 1. Experimental method

[0072] Take a 2.0mLEP tube and add 1mL soybean oil and 400μL TE buffer respectively. All the other steps are the same as in Example 1. Soybean seed DNA was extracted with A010301 Plant Genomic DNA Extraction Kit from APEXBIO Company as a control. Use the specific primers of the exogenous gene 35S, and use the DNA extracted by this method as a template to amplify by PCR to check whether the mentioned DNA is correct and whether it meets the requirements for transgenic detection.

[0073] 2. Experimental results

[0074] DNA was extracted from the transgenic soybean crude oil by the method described in Example 2, the concentration and purity of the DNA were determined, and the integrity of the DNA was detected by agarose gel electrophoresis. Experimental results show that the DNA extracted by this method meets the requirements of transgenic detecti...

Embodiment 3

[0077] Example 3 Using 1.5mL extraction system to extract DNA from Arowana brand transgenic soybean oil

[0078] 1. Experimental method

[0079] Take a 2.0mL EP tube and add 1mL Arowana soybean oil and 400μL TE buffer to each. All the other steps are the same as in Example 1. Soybean seed DNA was extracted with A010301 Plant Genomic DNA Extraction Kit from APEXBIO Company as a control. Use the specific primers of the exogenous gene 35S, and use the DNA extracted by this method as a template (see Table 6 for DNA concentration), and check whether the mentioned DNA is correct by PCR amplification. Whether it meets the requirements for genetically modified testing.

[0080] Table 6 Arowana brand soybean oil DNA extraction concentration list

[0081] sample number

260 / 280

Con(ng / μL)

s201508 Arowana brand soybean oil

1.16275

5.21653

[0082] 2. Experimental results

[0083] DNA was extracted from Arowana brand transgenic soybean oil by the m...

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Abstract

The invention provides a method for extracting DNA (deoxyribonucleic acid) from grain oil with low cost and low initial amount through an adsorption column. According to the method, DNA in the grain oil is extracted with water, a buffer solution or n-hexane in a 1.5 mL or 50 mL centrifuge tube through repeated cycling extraction, and then DNA is collected and purified by a centrifugal adsorption column. Relative concentration of extracted DNA is higher, and follow-up collection and purification are facilitated. The method is suitable for extracting DNA from the grain oil with low DNA content in experiments or detection centers of different scales. DNA extracted with the method can be subjected to PCR (polymerase chain reaction) amplification, genetically modified ingredient detection, species detection and the like.

Description

technical field [0001] The patent of the present invention relates to a low-cost, simple, convenient and rapid DNA extraction method from a small amount of grain and oil. Background technique [0002] With the maturity of transgenic technology, many crops have been commercialized, and the planting area has expanded rapidly. Genetically modified food is gradually on people's table, into people's food chain. Among the many plant raw materials used to manufacture and process genetically modified foods, soybeans, rapeseed, corn, potatoes, and tomatoes are the main ones. [0003] Because genetically modified foods contain new genetic material and proteins, these substances did not exist in traditional foods in the past. Therefore, the safety of genetically modified food has become one of the hot issues concerned by the international community and the general public. For genetically modified food, in order to protect human health, eliminate consumer concerns, and facilitate int...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/101C12Q2523/308
Inventor 龚建冯晓燕林挺王文利于祥春
Owner 北京爱普拜生物技术有限公司
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