实验试剂

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 × 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.

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 &mu;g). 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 &mu;g/&mu;l. 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 &mu;g 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 &mu;g, 6.5 &mu;g, and 5.8 &mu;g, respectively (2).
• Typical yield for animal tissues (&mu;g DNA/mg tissue): liver, kidney, or lungs, 3-5 &mu;g; skeletal muscle, heart, or brain, 1-3 &mu;g.
• 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 &mu;g/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).