实验概要

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

1. 100% ethanol
2. 75% ethanol
3. 8 N NaOH

实验步骤

Instructions for Use

Unless stated otherwise, the procedure is carried out at room temperature.

1. Lysis of cells and nuclei:

• 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.

• 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.

• Cell Nuclei: Add 1 ml of DNAZOL Reagent to 1-3 × 10⁷  cell nuclei, either in pellet or in suspension (volume < 0.1 ml).  Lyse the nuclei by inverting the assay tube or by gently pipetting the  mixture.

• To  minimize shearing of the DNA molecules, pipet DNA solution using  wide-bore pipette tips. Prepare wide bore pipette tips by cutting 2-3 mm  from the ends of plastic pipette tips. Mix DNA solutions by inversion;  avoid shaking or use of a Vortex for mixing.

Homogenization of tissues:

Homogenize tissue samples in a hand-held glass/Teflon^®  homogenizer. Use a loosely fitting homogenizer, with a tolerance of  0.1-0.15 mm or higher. Homogenize 25-50 mg tissue in 1 ml of DNAzol^®  Reagent by applying as few strokes as possible. Typically, 5-10 strokes  are required for complete homogenization. Small amounts (5-10 mg) of  soft tissues, such as spleen or brain, can be dispersed into smaller  fragments and lysed by repetitive pipetting with a micropipette. Plant  tissues may be efficiently powdered by first freezing in liquid nitrogen  or dry ice/ethanol before extraction with DNAzol^® Reagent.

2. Centrifugation (optional):

Sediment the homogenate for 10 min at 10,000 × g at 4°C or room  temperature. Following centrifugation, transfer the resulting viscous  supernatant to a fresh tube. This step removes insoluble tissue  fragments, RNA, and excess polysaccharides from the lysate/homogenate.  It is required only for the isolation of DNA from tissues such as liver,  muscles, and most plant tissues containing a large amount of cellular  and/or extracellular material. This process is recommended in order to  minimize RNA carry-over into the DNA.

3. DNA Precipitation:

Precipitate DNA from the lysate/homogenate by the addition of 0.5 ml of 100% ethanol per 1 ml of DNAzol^®  Reagent used for the isolation. Mix samples by inversion and store them  at room temperature for 1-3 min. DNA should quickly become visible as a  cloudy precipitate. Remove the DNA precipitate by spooling with a  pipette tip. Swirl the DNA onto the tip and attach it to the tube wall  near the top of the tube by gently sliding the DNA off the tip  (alternatively, transfer the DNA to a clean tube). Carefully decant the  supernatant, leaving the DNA pellet near the top of the tube. Place the  tubes upright for 1 min and aspirate the remaining lysate/homogenate  from the bottom of the tubes. If extensive pipetting is used to  facilitate lysis/homogenization before precipitation with ethanol, the  resulting sheared DNA will not spool. The same is true for small  quantities of DNA (< 15 μg). In this case, centrifugation at 4,000 × g  for 1-2 min at room temperature or 4°C will pellet the DNA.

4. DNA Wash:

Wash the DNA precipitate twice with 0.8-1.0 ml of 75% ethanol. At  each wash, suspend the DNA in ethanol by inverting the tubes 3-6 times.  Store the tubes vertically for 0.5-1 min to allow the DNA to settle to  the bottom of the tubes and remove ethanol by pipetting or decanting.

5. DNA Solubilization:

• Air  dry the DNA by storing in an open tube for 5-15 seconds after removing  the ethanol. (If the DNA is exposed to air for more than a few seconds,  it will be much more difficult to dissolve.) Dissolve the DNA in 8 mM  NaOH by slowly passing the pellet through a pipette tip. Use of the 8 mM  NaOH assures full solubilization of the DNA precipitate. Add an  adequate amount of the 8 mM NaOH to approach a DNA concentration of  0.2-0.3 μg/μl. Typically add 0.2-0.3 ml of 8 mM NaOH to the DNA isolated  from 10⁷ cells or 10-20 mg of animal tissue. DNA will not be  fully solubilized in TE or water. (The resolubilization of DNAZOL  –isolated DNA is low in Tris buffers. Therefore the use of 8 mM NaOH is  highly recommended.) DNA is stable in 8 mM NaOH for several months at  4°C and greater than one year at -20°C.

• The  DNA preparations isolated from tissues such as liver, muscles, and  plants may contain some insoluble material (mostly polysaccharides).  Remove the insoluble material by centrifugation at 12,000 × g for 10  min.

• Weak alkaline solutions are neutralized by CO₂ from the air. Once a month, prepare 8 mM NaOH from a 2-4 M NaOH stock solution that is less than six months old.

• After  DNA is solubilized in 8 mM NaOH, adjust the DNA solution to a desired  pH by the addition of HEPES. Use the following amounts of 0.1 M or 1 M  HEPES (free acid) per 1 ml of 8 mM NaOH:

6. Quantitation of DNA and Results:

• Mix an aliquot of solubilized DNA with 1 ml of 8 mM NaOH and measure A₂₆₀ and A₂₈₀ of the resulting solution. Calculate the DNA content assuming that one A₂₆₀ unit equals 50 μg of double-stranded DNA per ml.

• For calculations of a cell number in analyzed samples or an expected yield of DNA, assume that the amount of DNA per 10⁶ diploid cells of human, rat, and mouse origin equals 7.1 μg, 6.5 μg, and 5.8 μg, respectively (2).

• Typical yield for animal tissues (μg DNA/mg tissue): liver, kidney, or lungs, 3-5 μg; skeletal muscle, heart, or brain, 1-3 μg.

• The A₂₆₀/A₂₈₀  ratio of the isolated DNA is within the 1.6-1.9 range and with a  molecular weight ranging from 20 to 100 kb. The molecular weight of the  isolated DNA depends upon its shearing by mechanical forces applied  during lysis/homogenization or during solubilization of the DNA  precipitate.

• The  isolated DNA contains partially degraded RNA. If a reduction of the RNA  content to less than 3% is necessary, perform the centrifugation step  as described in Step 2 of the protocol. In Southern analysis, RNA can be  digested by supplementing the restriction mix with RNase A (1 μg/ml).

注意事项

The isolation procedure can be interrupted and samples can be stored as follows:

• The lysate/homogenate can be stored for 18 hours at 15 to 30°C or for three days at 2 to 8°C (refer to Step 1).

• During  washes, DNA can be stored in 95% ethanol for at least one week at 15 to  30°C or for three months at 2 to 8°C (refer to Step 4).