基因组DNA提取方法

cm2) of young field or greenhouse-grown plant leaves, filtered and dried mycelium, the muscle of one back leg of a grasshopper and shrimp muscle were used for DNA extraction. The fresh tissue was homogenized in 400 µl of sterile salt homogenizing buffer (0.4 M NaCl 10 mM Tris-HCl pH 8.0 and 2 mM EDTA pH 8.0), using a Polytron Tissue Homogenizer, for 10-15 s. Then 40 µl of 20% SDS (2% final concentration) and 8 µl of 20 mg/ml protenase K (400 µg/ml final concentration) were added and mixed well. The samples were incubated at 55-65°C for at least 1 h or overnight, after which 300 µl of 6 M NaCl (NaCl saturated H2O) was added to each sample. Samples were vortexed for 30 s at maximum speed, and tubes spun down for 30 min at 10 000 g. The supernatant was transferred to fresh tubes. An equal volume of isopropanol was added to each sample, mixed well, and samples were incubated at -20°C for 1 h. Samples were then centrifuged for 20 min, 4°C, at 10 000 g. The pellet was washed with 70% ethanol, dried and finally resuspended in 300-500 µl sterile dH2O. The purity of the DNA, determined from the A260/A280 ratio averaged >1.77 for all organisms. There was no RNA contamination in all samples nor any sign of degraded DNA during preparation (Fig. 1 ). The yield of DNA ranged from 500 to 800 ng/mg fresh weight for all individuals sampled. The amount of tissue required for this method is minimal, but we scaled up the amount of tissue 10-fold without any reduction in DNA quality and quantity. The average number of PCR reactions that can be performed using DNA extracted from 50 mg tissue was >3000. Universal and rapid salt-extraction of high quality genomic DNA for PCR- based techniques Nucleic Acids Res. 1997 25: 4692-4693http://nar.oupjournals.org/cgi/content/full/25/22/4692
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