SLS splitting liquor for rapid extraction of fungus DNA and uses thereof

A lysate and fungus technology, applied in the field of genetic engineering, can solve the problems of complex DNA method operation, prone to cross-contamination, difficult operation, etc., to simplify DNA extraction, ideal DNA concentration and purity, and avoid cross-contamination. Effect

Inactive Publication Date: 2008-07-30
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A large amount of DNA can be extracted at one time through these methods, but these methods have disadvantages such as time-consuming and laborious:
[0004] (2) After adding liquid nitrogen, strong grinding is required, and the actual operation is difficult;
[

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] (1) Use a sterile scalpel to scrape mycelia (about 50-200 mg, with a small amount of medium that does not affect DNA extraction) from the PDA plate and place it in a 2.0-mL Eppendorf tube, add 800 μL of SLS lysate (200 mmol / L Tris-HCl, 50mmol / L EDTA, 200mmol / L NaCl, 2g / 100ml sodium N-lauroyl sarcosinate, pH8.0), fully stir and disperse mycelia with a sterile toothpick, bathe in water at 55°C for 10min, shake and mix well 2-3 times, centrifuge at 13200r / min for 10min;

[0044] (2) Take 750 μL of the supernatant in a 1.5-mL Eppendorf tube, add an equal volume of chloroform and isoamyl alcohol (volume ratio 24:1) to extract DNA, and centrifuge at 13200 r / min for 10 min;

[0045] (3) Take the supernatant in a new 1.5-mL Eppendorf tube, add 2 times the volume of absolute ethanol to mix and mix, let stand at -20°C for 10 minutes, then centrifuge at 4°C and 13200r / min for 4 minutes to precipitate DNA;

[0046] (4) The precipitate was washed with 70% absolute ethanol, placed a...

Embodiment 2

[0048] (1) Scrape mycelia (about 50-200 mg) from the PDA plate with a sterile scalpel blade and place it in a 2.0-mL Eppendorf tube, add 800 μL SLS lysate (150 mmol / L Tris-HCl, 50 mmol / L EDTA, 150 mmol / L NaCl, 1g / 100ml (weight / volume) sodium N-lauroyl sarcosinate, pH8.0), fully stir and disperse mycelia with a sterile toothpick, bathe in 50°C water for 10min, shake and mix 2-3 times during the period, Centrifuge at 13000r / min for 10min;

[0049] (2) Take 500 μL of the supernatant in a 1.5-mL Eppendorf tube, add twice the volume of a mixed solvent of chloroform and isoamyl alcohol (volume ratio 24:1) to extract DNA, and centrifuge at 13000 r / min for 10 min;

[0050](3) Take the supernatant into a new 1.5-mL Eppendorf tube, add 3 times the volume of isopropanol to mix and mix, let stand at -20°C for 10 minutes, then centrifuge at 4°C and 13000r / min for 4 minutes to precipitate DNA;

[0051] (4) The precipitate was washed with 70% ethanol, placed at room temperature to dry natu...

Embodiment 3

[0053] (1) Use a sterile scalpel to scrape the mycelium (about 50-200 mg) from the PDA plate and place it in a 2.0-mL Eppendorf tube, add 800 μL of SLS lysate (300mmol / L Tris-HCl, 100mmol / L EDTA, 300mmol / L NaCl, 3g / 100ml (weight / volume) sodium N-lauroyl sarcosinate, pH7.5), fully stir and disperse mycelia with a sterile toothpick, bathe in 80°C water for 15min, shake and mix 2-3 times during the period, Centrifuge at 15000r / min for 10min;

[0054] (2) Take 750 μL of the supernatant in a 1.5-mL Eppendorf tube, add an equal volume of mixed solvent of phenol, chloroform and isoamyl alcohol (volume ratio 25:24:1) to extract DNA, and centrifuge at 15000 r / min for 10 min;

[0055] (3) Take the supernatant into a new 1.5-mL Eppendorf tube, add 2 times the volume of isopropanol to mix and mix, let stand at -20°C for 10 minutes, then centrifuge at 4°C, 15000r / min for 4 minutes to precipitate DNA;

[0056] (4) The precipitate was washed with 75% ethanol, placed at room temperature to ...

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PUM

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Abstract

The invention discloses a SLS lysis solution which can quickly extract fungal DNA. The SLS lysis solution contains following components (calculated by the total volume of solution): 1 - 3 g/100ml of N-lauroyl sodium sarcosinate, 150 - 300mmol / L of Tris-HCl, 150 - 300mmol / L of NaCl, and 50 - 100mmol / L of EDTA; and the pH value is ranged from 7.5 to 8.5. The SLS lysis solution of the invention takes N-lauroyl sodium sarcosinate as the main functional component to make up the DNA extracting solution; and the DNA extracting solution can be used for extracting fungal DNA. Due to the excellent biologic degradability, the DNA extracting solution can be used for directly breaking the cell wall of pathogenic fungi, thereby avoiding the complicated physical processing of the cell-wall breaking in traditional method in extracting fungal DNA, and greatly simplifying the extracting of DAN.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an SLS lysate for rapidly extracting fungal DNA and its application. Background technique [0002] Common fungal DNA extraction method has CTAB extraction method (Saghai, M.A.K.M.Soliman, R.A.Jorgensen&R.W Allard, 1984, Ribosomal DNA spacer-length polymorphism in barley:Mendelian inheritance, chromosomal location and population dynamics.Prov.Natl.Acad.Sci.USA, 81(24):8014-8018.), urea extraction method (Sun, Y, W. Zhang, F. Li, Y. Guo, T. Liu & H. Huang, 2000, Identification and genetic mapping of four novel genes that regulate leaf development in Arabidopsis. Cell Research, 10(4); 325-335.), SDS extraction method (He, Y.Q. 2000, An improved protocol for fungal DNA preparation. Mycosystema, 19(3): 434.), etc. The main technical routes of these methods are: using liquid medium to cultivate fungi, collecting mycelium, grinding and breaking the cell wall with liquid nit...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 陈广进李红叶张志芳
Owner ZHEJIANG UNIV
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