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Method for extracting sedum spectabile genomic DNA

An extraction method and genome technology, applied in the field of Sedum sedum genomic DNA extraction, can solve the problems of complicated operation and low quality of genomic DNA, and achieve the effects of improving purity, reducing pollution and ensuring reliability

Inactive Publication Date: 2013-01-16
TIANJIN CITY AGRI BIO TECH RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some methods can obtain genomic DNA, the quality of the extracted genomic DNA is not high, and the whole operation is complicated

Method used

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  • Method for extracting sedum spectabile genomic DNA
  • Method for extracting sedum spectabile genomic DNA
  • Method for extracting sedum spectabile genomic DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] (1) Take 0.2g of fresh leaves and put them into a mortar, add a small amount (0.02g) of quartz sand, fully grind them into powder with liquid nitrogen, add 0.5mL of preheated 2% CTAB mixed extract solution to dissolve, 2% CTAB mixed extract The solution includes: 2% CTAB, 2% SDS, 2% PVP, 0.1mol / L Tris-Cl, 20mmol / L EDTA, 1.4mol / L NaCl and 10% β-mercaptoethanol, the Tris-Cl The pH value is 8.0, put into a 1.5mL centrifuge tube;

[0045] (2) Put it in a water bath for 45 minutes at 60°C, take it out and add about 0.5mL of chloroform-isoamyl alcohol mixture after cooling, and mix it. The ratio of chloroform and isoamyl alcohol in the chloroform-isoamyl alcohol mixture is: 24:1, centrifuge at 12000 rpm for 10 minutes at room temperature;

[0046] (3) Take the supernatant and transfer it to a centrifuge tube, then add an equal volume of chloroform-isoamyl alcohol mixture (the ratio of chloroform to isoamyl alcohol is 24:1) and mix. minute;

[0047] (4) Transfer the superna...

Embodiment 2

[0050] Extraction method of Genomic DNA of Babao Sedum:

[0051] (1) Put 0.2g of fresh leaves into a mortar, add 0.04g of quartz sand, fully grind into powder with liquid nitrogen, add 0.5mL of preheated 2% CTAB extract to dissolve;

[0052] The 2% CTAB extract is composed of the following raw materials:

[0053] 2% CTAB 2g / mL

[0054] 2%SDS 2g / 100mL

[0055] 2%PVP 2g / 100mL

[0056] 0.1mol / LTris-Cl 1.1214g / 100mL with a pH value of 8.0

[0057] 20mmol / LEDTA0.744g / 100mL

[0058] 1.4mol / LNaCl 8.19g / 100mL

[0059] 10% β-mercaptoethanol 10ml / 100mL.

[0060] (2) Incubate at 55°C for 45 minutes, take it out and cool it, then add 1mL of chloroform-isoamyl alcohol mixture, centrifuge at 12000-15000 rpm for 10 minutes at room temperature; the volume ratio of chloroform to isoamyl alcohol is 24: 1;

[0061] (3) Take the supernatant and transfer it to a new centrifuge tube, then add an equal volume of chloroform-isoamyl alcohol mixture (the volume ratio of chloroform to isoamyl a...

Embodiment 1、2

[0064] Embodiment 1, 2 gained sedum sedum genome DNA detection analysis:

[0065] In order to compare the quality of DNA extracted by different methods, 2 μL DNA solutions were taken for gel electrophoresis analysis. 1% agarose (Spain) gel, 1×TAE (Shanghai Bioengineering Company), Goldview (Beijing Saibaisheng Gene Technology Co., Ltd.) staining, the loaded gel was placed in the electrophoresis tank (JUNYI JY-SP3, Beijing ) to detect DNA by electrophoresis at 110V for 15 minutes. Scanning and imaging were performed using the SIM UV gel imaging system (Bio-best, USA). Such as image 3 As shown, after the DNA extracted by the above-mentioned embodiments is detected by electrophoresis, clear bands can be observed, and it can be seen that the Sedum sedum DNA extracted from the above-mentioned embodiments has a large content and high purity.

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Abstract

The invention discloses a method for extracting sedum spectabile genomic DNA, comprising the following steps: taking powdered sedum spectabile, and adding preheated 2% CTAB extracting solution to dissolve; carrying out warm bath at 65 DEG C for 45 minutes, adding hloroform and isoamyl alcohol mixture of same volume, mixing, and centrifuging for two times at high speed at room temperature; taking a supernatant, adding ice-cold isopropanol which is 2 / 3 the volume of the supernatant, mixing, standing at low temperature for 15-30 minutes, and settling DNA; centrifuging at high speed at low temperature, discarding the supernatant, recovering the precipitate, washing for two times by 70% ethanol, airing, adding TE buffering solution, adding RNase, digesting for 30 minutes at 37 DEG C, and preserving the obtained DNA sample at low temperature. According to the method, the added RNase which is an enzyme can remove the RNA thoroughly and effectively so that the extracted DNA has higher purity and the interference of the RNA to the subsequent experiment is avoided.

Description

technical field [0001] The invention relates to a technology for separating and extracting plant genome DNA in molecular biology experiments, in particular to a method for extracting genome DNA of Sedum babao. Background technique [0002] Babao Sedum ( Sedum spectabile ) is a succulent herb of the sedum family Sedum. It likes strong light, is resistant to barrenness and drought, and can withstand low temperatures of -25°C. It is extensively managed and has few pests and diseases. It is a rare and excellent landscaping material. As a landscape project, the group greening effect of Babao Sedum is excellent, and it is also a good pattern material. At the same time, the whole herb can be used as medicine, which has the effects of dispelling wind and dampness, promoting blood circulation and dissipating blood stasis, and stopping bleeding and relieving pain. Sedum babao contains rich polysaccharides and other secondary metabolites, which makes it have a high utilization value,...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 周祥明宋建王姝
Owner TIANJIN CITY AGRI BIO TECH RES CENT
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