Extraction of DNA using DNAzol Reagent
互联网
1272
实验概要
Isolation of genomic DNA from solid and liquid samples of animal, plant, yeast, and bacterial origin.
实验原理
DNAzol® Reagent (Genomic DNA Isolation Reagent) is a complete and ready-to-use reagent for the isolation of genomic DNA from solid and liquid samples of animal, plant, yeast, and bacterial origin. The DNAzol® Reagent procedure is based on the use of a novel guanidine-detergent lysing solution which permits selective precipitation of DNA from a cell lysate. Since first proposed by Cox (1), the isolation of genomic DNA with guanidine salts has been the subject of numerous reports and commercial applications. Developed by Chomczynski, DNAzol® Reagent is an advanced DNA isolation reagent that combines both reliability and efficiency with simplicity of the isolation protocol. The DNAzol® Reagent protocol is fast and permits isolation of genomic DNA from a large number of samples of small or large volumes.
During the isolation, a biological sample is lysed (or homogenized) in DNAzol® Reagent and the genomic DNA is precipitated from the lysate with ethanol. Following an ethanol wash, DNA is solubilized in water or 8 mM NaOH. The procedure can be completed in 10- 30 min with DNA recovery of 70-100%. The isolated DNA can be used without additional purification for applications such as Southern analysis, dot blot hybridization, molecular cloning, and polymerase chain reaction (PCR).
主要试剂
DNAzol® Reagent
100% ethanol
75% ethanol
8 N NaOH
实验步骤
1 Lysis of cells and nuclei:
1) Cells grown in monolayer: Add 0.75-1.0 ml of DNAzol® Reagent per 10 cm2 culture plate area. Lyse the cells by agitating the culture plate and gently pipet the lysate into an assay tube.
2) Cell Pellets or Suspensions: Add 1 ml of DNAzol® Reagent to 1-3 × 107 cells, either in pellet or in suspension (volume < 0.1 ml). Lyse the cells by gently pipetting. For whole blood up to 100 µl, add 1 ml of DNAZOL to the blood and pipet up and down gently to lyse the cells. For whole blood (>100 µl), pellet the cells and wash them with 0.9% NaCl. Pellet the cells again and resuspend them in one volume of cold (4°C) hypotonic solution [20 mM Tris HCl (pH 8.0), 10 mM EDTA]. Pellet the cells at 4,000 rpm for 10 min (4°C). Discard the supernatant and add 1 ml DNAzol® per 1-3 × 107cells. Lyse the cells by gently pipetting.
Isolation of genomic DNA from solid and liquid samples of animal, plant, yeast, and bacterial origin.
实验原理
DNAzol® Reagent (Genomic DNA Isolation Reagent) is a complete and ready-to-use reagent for the isolation of genomic DNA from solid and liquid samples of animal, plant, yeast, and bacterial origin. The DNAzol® Reagent procedure is based on the use of a novel guanidine-detergent lysing solution which permits selective precipitation of DNA from a cell lysate. Since first proposed by Cox (1), the isolation of genomic DNA with guanidine salts has been the subject of numerous reports and commercial applications. Developed by Chomczynski, DNAzol® Reagent is an advanced DNA isolation reagent that combines both reliability and efficiency with simplicity of the isolation protocol. The DNAzol® Reagent protocol is fast and permits isolation of genomic DNA from a large number of samples of small or large volumes.
During the isolation, a biological sample is lysed (or homogenized) in DNAzol® Reagent and the genomic DNA is precipitated from the lysate with ethanol. Following an ethanol wash, DNA is solubilized in water or 8 mM NaOH. The procedure can be completed in 10- 30 min with DNA recovery of 70-100%. The isolated DNA can be used without additional purification for applications such as Southern analysis, dot blot hybridization, molecular cloning, and polymerase chain reaction (PCR).
主要试剂
DNAzol® Reagent
100% ethanol
75% ethanol
8 N NaOH
实验步骤
1 Lysis of cells and nuclei:
1) Cells grown in monolayer: Add 0.75-1.0 ml of DNAzol® Reagent per 10 cm2 culture plate area. Lyse the cells by agitating the culture plate and gently pipet the lysate into an assay tube.
2) Cell Pellets or Suspensions: Add 1 ml of DNAzol® Reagent to 1-3 × 107 cells, either in pellet or in suspension (volume < 0.1 ml). Lyse the cells by gently pipetting. For whole blood up to 100 µl, add 1 ml of DNAZOL to the blood and pipet up and down gently to lyse the cells. For whole blood (>100 µl), pellet the cells and wash them with 0.9% NaCl. Pellet the cells again and resuspend them in one volume of cold (4°C) hypotonic solution [20 mM Tris HCl (pH 8.0), 10 mM EDTA]. Pellet the cells at 4,000 rpm for 10 min (4°C). Discard the supernatant and add 1 ml DNAzol® per 1-3 × 107cells. Lyse the cells by gently pipetting.
