农杆菌质粒DNA高效提取方法研究

以EHA105菌株为实验材料,通过优化质粒扩增条件、加大菌液使用量、改良提取纯化过程中所用试剂等,简化了操作流程,给出了一种高效提取农杆菌中质粒DNA的方法。结果表明,选择LB＋KANA为培养基、抗生素质量浓度为50 mg/L,细菌培养至OD600=0.634～0.714时,以5 mL/次用量取菌液裂解;以改进方法提取试剂,得率与常规方法相当。该方法在满足PCR检测条件的前提下既减少了酚、氯仿等有毒试剂的用量,又省时、经济、方便,为植物转基因工程中目的物DNA的检测提供了技术支持。

Research on a Method of Efficient Extraction of Plasmid DNA from Agrobacterium

The establishment of the method in which the plasmid DNA can be extracted efficiently from Agrobacterium provides technical support for the test of the target DNA in the plants＇ transgenic engineering. The extraction method is acquired based on a series of experiments with EHA105 Agrobacterium as material, which is carried out by optimizing the conditions of plasmid amplification, increasing the amount of bacterial liquid, improving the reagents in the process of extraction and purification and simplifying the operational processes. The result shows that taking LB ＋ KANA as the medium, when concentration of antibiotic is 50 mg/L, bacteria are cultured to OD600=0.634 - 0.714; taking 5 mL bacterial liquid every time, the bacteria is cracking. The dosage of reagent used in the extraction is referred to in the improved methods; yield is consistent with the conventional method. Meeting PCR detection, the improved methods not only decrease the use of toxic reagents such as phenol, chloroform etc, but also are more time-saving, more economical, and more convenient. The method establishes the foundation for its wide application to ordinary experiments.