可转化人工染色体(TAC)文库载体DNA的制备

Preparation of the Vector DNA for Library from the Transformation-competent Artificial Chromosome

对可转化人工染色体(TAC)pYLTAC747NH/sacB文库载体DNA的制备条件进行了较系统的摸索和研究。结果表明,该文库载体经碱裂解法提取、QIAGEN Plasmid Mini kit纯化后,可获得较纯净的载体DNA。对其闭环载体DNA分别用不同酶量的Hind III酶切处理,经琼脂糖凝胶电泳检测得出其最佳Hind III完全酶切条件为2 U Hind III/μg闭环载体DNA、37℃酶切30 min;分别用0.5 MBU和1 MBU HK脱磷酶/μg对其线性载体DNA进行脱磷处理,经电泳和载体自连产物电转化检测表明其适宜的完全脱磷条件为1 MBU HK脱磷酶/μg线性载体DNA,30℃脱磷1 h;将所制备的线性载体DNA与λDNA/Hind III酶切片段进行连接,连接产物转化频率较高,其电转化大肠杆菌DH10B感受态细胞频率可达到9.6×108。

The optimal condition for the preparation of qualified vector DNA from the transformation-competent artificial chromosome（TAC）pYLTAC747NH/sacB for library was groped and studied.The result showed that the pure vector DNA could be obtained after the plamid was extracted by the alkaline lysis method and purified by QIAGEN Plasmid Mini kit.The closed-loop vector DNA was digested by gradient units of Hind III respectively.The result examined by agarose gel electrophoresis indicated that the optimal digestion conditions by Hind III was 2 U Hind III/μg vector DNA at 37℃ for 30 min.Subsequently,the linear vector DNA is dephosphorized by 0.5 MBU and 1 MBU HK thermolabile phosphatase respectively.The results determined by agarose gel electrophoresis,self-ligation reaction of the vector and eletroporation showed the suitable conditions for the complete dephosphorylation was 1MBU HK thermolabile phosphatase /μg linear vector DNA at 30℃ for 1 h.The vector DNA prepared and the DNA fragments from λ DNA/HindIII were ligated,and the ligation product were eletroporated into E.coli DH10B electrocompetent cells after spotdialysis.The transformation frequency was pleasing,which could come up to 9.6×108.