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目的探索一种适用于虫草属基因组DNA快速提取的方法。方法在无液氮条件下使用碱裂解法提取虫草DNA,使用ITS通用引物进行PCR扩增。考察了提取液配方、煮沸时间对DNA提取的影响以及中和剂Tris-HCl用量对PCR扩增的影响。利用优化的提取方法提取了虫草属5种虫草并用ITS通用引物进行PCR扩增。结果 80μl 0.5 mol/L Na OH进行提取,加入160μl 0.1 mol/L Tris-HCl中和,离心即可在10min内提取出虫草属真菌的DNA,PCR扩增能获得清晰目的条带。结论优化的碱裂解法可用于虫草属DNA的快速提取。
Objective To explore a method suitable for rapid genomic DNA extraction from Cordyceps. Methods Cordyceps militaris DNA was extracted by alkaline lysis without liquid nitrogen, and ITS universal primers were used for PCR amplification. The effects of the extract formulation, boiling time on DNA extraction and the amount of Tris-HCl neutralizer on the PCR amplification were investigated. Five kinds of Cordyceps sinensis were extracted by optimized extraction method and amplified with ITS universal primers. Results 80 μl of 0.5 mol / L NaOH was used for extraction. After 160 μl of 0.1 mol / L Tris-HCl was neutralized, the DNA of Cordyceps was extracted in 10 min by centrifugation. Conclusion The optimized alkaline lysis method can be used for rapid DNA extraction from Cordyceps.