ExtractionofDNAFromPlantsUsingPlantDNAzol?Reagent

實驗概要

Plant DNAzol^? 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^?. 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.

主要試劑

1. Ethanol
2. TE buffer (pH 8.0) or 8 mM NaOH
3. Chloroform.

實驗步驟

Protocol Summary

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 DNAzo^?. (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.

• For procedures such as PCR which require limited amounts of DNA, addition of chloroform is optional.

• Protocol  is written for isolation of DNA from 0.1 g of plant tissue. For larger  amounts of plant tissue, scale up volume of reagents proportionately.

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.

• When processing large samples (>0.5 g) disperse the DNA pellet in the wash solution with a transfer pipette.

• The volume of wash solution equals the volume of DNAzol^?  used for the original extraction. When processing plant material with a  small amount of contaminants, the volume of the wash solution can be  decreased by 50%.

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.

• For  large samples (>0.5 g), an additional ethanol wash might be  necessary to remove chlorophyll and other pigments from the DNA pellet.

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.

• Typical  yield is 50 - 300 μg of DNA/g of plant leaf material. Add an adequate  amount of TE buffer (pH 8.0) or 8 mM NaOH to achieve a DNA concentration  of 0.1 - 0.3 μg/μl.

• Genomic  DNA is difficult to solubilize and repeated pipetting is required for  its complete solubilization. Incomplete solubilization will result in  loss of DNA during the final centrifugation step.

• 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 6 months old.

• If  DNA 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 ml of 8 mM NaOH:

Quantitation of DNA And Results:

• Mix an aliquot of the solubilized DNA with 1 ml of TE buffer (pH 8.0) or 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/ml (2).

• Molecular weight of the isolated DNA ranges from 20 to 100 kb with the A₂₆₀/A₂₈₀  ratio ranging 1.6 - 1.9. The molecular weight of the isolated DNA is  influenced by the extent of DNA shearing during tissue grinding.

注意事項

1. Plant DNAzol^?  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.
2. 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^?).

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

• Plant DNAzol^?  extract, before or after the initial centrifugation step (step 1.2),  can be stored for at least one week at room temperature, and at least  one month or one year at 4°C or -20°C, respectively.

• The DNA pellet can be stored in 95% ethanol for at least one week at room temperature or for one year at 4°C.