高效制备双元细菌人工染色体(BIBAC)载体DNA的研究

Research on Efficient Preparation of Binary Bacterial Artificial Chromosome (BIBAC) Vector DNA

高质量的BIBAC载体DNA的制备受到酶切、脱磷等许多因素的影响,是构建BIBAC文库的关键步骤之一.以BIBAC2载体为材料,对其进行了BamHI限制性酶切和CIAP脱磷,制备了可用于进一步构建文库的线性载体.在此基础上确定了适宜的酶量、酶切时间、脱磷酶的用量及失活等步骤的反应条件.

Preparation of high purified vector DNA, which is a key step in BIBAC library construction, is affected by a series of factors including digestion of restriction enzyme and dephosphorylation of linearized vector DNA. In our study, high quality of vector DNA for construction of BAC library was prepared after the BIBAC vector was digested by the restriction enzyme of BamH I and dephosphorylation by CIAP with the modified conditions including optimal concentration of restriction enzyme, optimal digestion time, optimal concentration of dephosphatase and optimal treatment of dephosphatase inactivity.