BMBMethod:ArabiDNA

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One-step DNA extraction from Arabidopsis thaliana

Overview
To lessen labor, time or cost of DNA extraction, we established a one-step method of DNA extraction from Arabidopsis. After several trials, we found that a diluted extraction buffer from a known protocol (Edwards et al. 1991) can be successfully used to extract DNA in one step. This is one of the one-step protocols for DNA extraction from Arabidopsis.

Notes
This protocol is aimed for the extraction of DNA that is used as PCR template, and not for DNA isolation per se. Additionally, experiments might not be successful when the ratio of plant sample to extraction buffer is greatly changed, plant tissue is vigorously crushed with machines, or the volume of extract added to PCR reaction is increased.

Preparation
Edwards Solution (200 mM Tris-HCl (pH 7.5), 250 mM NaCl, 25 mM EDTA, and 0.5% SDS)
TE Buffer (10 mM Tris-HCl (pH 8) and 1 mM EDTA)
(Dilute Edwards Solution by 10-fold with TE Buffer to obtain Extraction Buffer)
Leaf sample (3–5 mg)
1.5 mL plastic tube
Plastic rods that fit the tube (Wash and sterilize with autoclave)

Protocol
Place leaf sample in plastic tube. Add 200 μL Extraction Buffer to the tube. Crush leaf with plastic rod against the tube wall. The solution turns transparent green, and visible tissue residue is left in the solution. This solution can be successfully used in the following PCR reaction. If you want to remove tissue residue, centrifuge tube at 14,000 rpm for 5 min and recover supernatant. This solution is stable at -20oC for several months with or without tissue residue. Add 1 μL of this solution to in total 20 μL of PCR reaction. Addition of 2 μL inhibits PCR reaction.

References

Kasajima I., Ide Y., Ohkama-Ohtsu N., Yoneyama T., Fujiwara T. (2004) A protocol for rapid DNA extraction from Arabidopsis thaliana for PCR analysis. Plant Mol. Biol. Rep. 22, 49-52
Edwards K., Johnstone C., Thompson C. (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucl. Acid. Res. 19: 1349