Extraction of DNA using DNAzol® Reagent
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1047
实验试剂
1. 100% ethanol
2. 75% ethanol
3. 8 N NaOH
实验步骤
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1 ml DNAzol® Reagent 25-50 mg tissue 1 - 3x107 cells, 0.1 ml liquid sample |
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Unless stated otherwise, the procedure is carried out at room temperature.
- 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 × 107 cells. 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.
- 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:
