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Extraction of DNA From Plants |
Plant DNAzol™ Reagent is an extra-strength-DNAzol reagent (patent pending) specifically formulated for the isolation of genomic DNA from plants. The Plant DNAzol procedure is based on the use of a novel guanidine-detergent lysing solution which hydrolyzes RNA and allows the selective precipitation of DNA from the lysate. The Plant DNAzol protocol is fast and permits efficient isolation of genomic DNA from a variety of plant tissues.
In the Plant DNAzol procedure, plant samples are pulverized in liquid nitrogen or homogenized, and genomic DNA is extracted from the homogenate with Plant DNAzol Reagent. Following extraction, plant debris is removed by centrifugation and DNA is precipitated from the supernatant with ethanol. The resulting DNA pellet is washed with ethanol and solubilized. The entire procedure can be completed in ~60 min and the isolated DNA can be used for Southern analysis, dot blot hybridization, molecular cloning, PCR, molecular mapping, and other biology and biotechnology applications.
Stability:
Plant DNAzol Reagent is stable at room temperature for at least one year after the date of purchase.
Handling precautions:
Plant DNAzol Reagent contains irritants. Handle with care, avoid contact with skin, use eye protection (shield, safety goggles). In case of contact, wash skin with a copious amount of water; seek medical attention.
Protocol summary
1.
|
Extraction
|
0.3 ml Plant DNAZOL + 0.1 g pulverized plant tissue: 0.3 ml chloroform
|
12,000 ×
g, 10 min
|
2.
|
DNA precipitation
|
supernatant + 0.225 ml 100% ethanol
|
5,000 ×
g, 4 min
|
3.
|
DNA wash
|
0.3 ml Plant DNAZOL-ethanol solution
0.3 ml 75% ethanol
|
5,000 ×
g, 4 min
5,000 ×
g, 4 min
|
4.
|
DNA solubilization
|
TE buffer (pH 8) or 8 mM NaOH
|
12,000 ×
g, 4 min
|
The procedure is carried out at room temperature. Centrifugation can be performed at 4°C to 25°C.
1. Extraction:
Pulverize plant tissue in liquid nitrogen using a mortar and pestle. Replenish the liquid nitrogen in the mortar 2 to 3 times and continue to grind sample until a fine, homogenous powder is obtained. Using a spatula, transfer the frozen powder to a microcentrifuge tube containing Plant DNAzol. (Use 0.3 ml Plant DNAzol for 0.1 g of plant tissue.) Mix the solution thoroughly by gentle inversion a few times and incubate at 25°C with shaking for 5 min. Add 0.3 ml chloroform, mix vigorously, and further incubate at 25°C with shaking for another 5 min. Centrifuge as described below (1.,2).
Following extraction, centrifuge the extracts at 12,000 × g for 10 min and transfer the resulting supernatant, or the aqueous phase after the chloroform extraction, to a fresh tube.
2. DNA precipitation:
Following centrifugation, precipitate DNA by mixing the aqueous phase with 0.225 ml of 100% ethanol. After addition of ethanol (2.1), mix samples by inverting the tubes 6 to 8 times and store them at room temperature for 5 min. Sediment precipitated DNA at 5,000 × g for 4 min, and remove the resulting supernatant. In some samples, DNA precipitate is not visible before centrifugation.
3. DNA wash:
Plant DNAzol-ethanol wash
Prepare Plant DNAzol-ethanol wash mixture by mixing 1 volume of Plant DNAZOL with 0.75 volume of 100% ethanol. Mix 0.3 ml of Plant DNAzol -ethanol wash solution with the DNA precipitate by vortexing. Store samples for 5 min and centrifuge at 5,000 × g for 4 min.
Ethanol wash
Remove the DNAzol wash solution, and wash the DNA pellet by vigorous mixing with 0.3 ml of 75% ethanol followed by centrifugation at 5,000 × g for 4 min.
4. DNA Solubilization:
Remove the ethanol wash by decanting, store tubes vertically for 1-2 min and remove the remaining ethanol with a micropipette. Air dry the DNA pellet. Dissolve the DNA pellet in 70 µl TE buffer (pH 8.0). If the DNA pellet is difficult to dissolve, use 8 mM NaOH instead of TE buffer. In a typical DNA preparation, the DNA solution is cloudy and may contain insoluble material. This insoluble material is removed by centrifugation at 12,000 × g for 4 min.
Final pH | 0.1 M HEPES (µl) | Final pH | 1.0 M HEPES(µl) |
---|---|---|---|
8.4 | 86 | 7.2 | 23 |
8.2 | 93 | 7.0 | 32 |
8.0 | 101 | ||
7.8 | 117 | ||
7.5 | 159 |
Quantitation of DNA And results:
Notes:
1. The isolated DNA may contain degraded RNA. To avoid RNA contamination, add RNase to Plant DNAzol at the beginning of the isolation procedure (100 µg RNase A/ml Plant DNAzol).
2. The isolation procedure can be interrupted and samples can be stored as follows:
仅供科研使用,不可用于诊断目的。