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Application of Guanidine Isothiocyanate |593-84-0| in Extraction of DNA and RNA

2015-09-11 来源:亚科官网
Abstract: This paper describes the use of guanidinium isothiocyanate, which is the simultaneous extraction of animal DNA virus and RNA virus nucleic acids in serum and double swabs. The method combines the classical Trizol extraction method and the silica gel column extraction method using Trizol as the lysate to make up for the defects of the two methods.
Key words: guanidinium isothiocyanate method, 593-84-0, extraction of DNA and RNA, Trizol extraction, silica gel column extraction
Foreword
Guanidine isothiocyanate, CAS No.: 593-84-0, Molecular formula: C2H6N4S, molecular weight: 118.161, white crystalline powder, melting point 115-120°C, used for denaturing lysing cells and extraction of RNA and DNA. It has no RNase and DNase action, and it is a strong protein denaturing agent. The article describes the patented isothiocyanate extraction method for simultaneous extraction of DNA viruses and RNA viral nucleic acids.
Simultaneous extraction of DNA virus and RNA virus nucleic acid by guanidinium isothiocyanate
With the rapid development of molecular biology, genetic diagnosis has become the most accurate and effective means to detect animal diseases. In the process of genetic diagnosis, the extraction of viral nucleic acids is a prerequisite for detection. The quality of nucleic acid extraction also affects the accuracy of detection. .
At present, animal virus nucleic acid extraction methods mainly include classical Trizol extraction method and silica gel column extraction method using Trizol as lysate. The classical Trizol extraction process is cumbersome, long cycle, low extraction efficiency and high technical requirements. The silica gel column extraction method using Trizol as lysate is costly, the quality of RNA extraction is not high, and only RNA virus can be extracted.
A method for simultaneously extracting animal DNA virus and RNA virus nucleic acid in serum and double swab, comprising the steps of: lysing the extract with guanidinium isothiocyanate lysate; adsorbing RNA by silica gel membrane; removing impurity protein with washing liquid I; wash away impurities with washing liquid II; DEPC water elutes nucleic acid; the above guanidinium isothiocyanate lysate comprises 3~7M guanidinium isothiocyanate, 0.6%~1.0% TriTon-100, 30~50 mM Tris-Cl, 5~15 mM DTT, 60~90 μg/mL proteinase K, 10~30 mM EDTA, pH 4.3~4.6.
The above lotion I includes 5~6M guanidine hydrochloride, 53%~59% absolute ethanol, K70~90 μg/mL protease, and the pH is 6.4~6.6; the above lotion II includes 70~80% ethanol.
Further, the above guanidinium isothiocyanate lysate comprises 5M guanidinium isothiocyanate, 0.8% TriTon-100, 40mM Tris-Cl, 10mM DTT, 80μg/mL proteinase K, 20mM EDTA, and the pH is 4.5. Further, the above lotion I comprises 5.5M guanidine hydrochloride, 56% absolute ethanol, 80μg/mL proteinase K, and a pH of 6.5. Further, the above lotion II includes 75% ethanol.
The above steps include:
(1) Take a clean centrifuge tube, add 100μL of the sample to 200μL of guanidinium isothiocyanate lysate, mix by shaking, and stand for 5 minutes.
(2) An equal volume of absolute ethanol, i.e., 200 μL, was added to the guanidinium isothiocyanate lysate, and the mixture was repeatedly mixed for 15 seconds.
(3) The filter column was placed on a 2mL collection tube, and the entire mixture was transferred to a filter column and centrifuged at 12,000 rpm for 1 minute.
(4) Discard the liquid in the collection tube, add 600μL of the washing solution 1 to the filter column, and centrifuge at 12,000 rpm for 1 minute.
(5) Discard the liquid in the collection tube, add 600 μL of the washing solution 2 to the filter column, centrifuge at 12,000 rpm for 1 minute, and repeat.
(7) discarding the liquid in the collection tube, the filter column is placed on the collection tube, and centrifuged at 12000 rpm for 1 minute;
(8) Replace the filter column with a new 1.5 mL centrifuge tube, add 20 μL of DEPC to the filter membrane, leave it at room temperature for 1 minute, centrifuge at 12,000 rpm for 1 minute, collect the filtrate, and store.
Conclusion
The invention has the beneficial effects of simultaneously extracting the animal RNA virus and the DNA virus nucleic acid in the serum and double swab sample, and has the characteristics of simple, rapid and low cost. This method lays the foundation for the establishment of nucleic acid extraction of animal RNA virus and DNA virus multiplex PCR methods.
References
Zhao Hongbo; Chen Bangzhao, A method for simultaneously extracting animal DNA virus and RNA virus nucleic acid from serum and double swab [P], CN103614371A, 2014.03.05
Related article: Product quality inspection specification of guanidinium isothiocyanate|593-84-0 |
Edited by Suzhou Yacoo Science Co., Ltd.