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1， 贴壁细胞用胰酶消化，离心收集。
2， 细胞重悬于冰冷的PBS漂洗一次，离心收集。
3， 重复2。
4， 加入5ml DNA提取缓冲液，(10m mol/L Tris-cl, 0.1 mol/L EDTA, o.5% SDS),混匀。
5， 加入25ul 蛋白酶K ,使终浓度达到100ug/ml, 混匀，50℃水浴3h,
6， 用等体积的酚抽提一次，2500r/min 离心收集水相，
用等体积的（酚，氯仿，异戊醇）混合物抽提一次，2500r/min 离心收集水相。
用等体积的氯仿，异戊醇抽提一次。
7， 加入等体积的5mol/L 的LiCL,混匀，冰浴，10min.
8， 2500r/min,离心10min. 转上清与一离心管中。加入等体积的异丙醇。室温10分钟。
9， 2500r/min,离心10min。弃上清。加入0.1倍 体积3mol/L 乙酸钠(PH5.2)与2倍体积-20℃预冷无水乙醇。-20℃ 20分钟。
10， 12000r/min, 室温离心5分钟。弃上清。将DNA溶于适量TE中。
About 50-100 mg (1 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： 4692-4693
http：//nar.oupjournals.org/cgi/content/full/25/22/4692