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Search Thermo Fisher Scientific
Extraction of DNA Using DNAzol Reagent |
Invitrogen 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 an easy 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).
Stability:
DNAzol Reagent is stable at 15 to 30°C for at least one year after the date of purchase.
Reagents and solutions
Instructions for use
1. Lysis/Homogenization | 1 ml DNAzol Reagent + 25-50 mg tissue 1 - 3x107 cells, 0.1 ml liquid sample |
2. Centrifugation (optional) | 10,000 × g, 10 min |
3. DNA Precipitation | Lysate + 0.5 ml 100% ethanol |
4. DNA Wash | 1 ml 75% ethanol (2X) |
5. DNA Solubilization | 8 mM NaOH ethanol and 8 mM NaOH. |
Unless stated otherwise, the procedure is carried out at room temperature.
1. Lysis of cells and nuclei:
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:
Final pH | 0.1 M HEPES (μl) | Final pH | 1 M HEPES (μl) |
8.4 | 86 | 7.2 | 23 |
8.2 | 93 | ||
8.0 | 101 | ||
7.8 | 117 | ||
7.5 | 159 |
6. Quantitation of DNA and results:
Notes:
The isolation procedure can be interrupted and samples can be stored as follows:
For Research Use Only. Not for use in diagnostic procedures.