Research On The Preparation Of Vector DNA About Transformation-competent Artificial Chromosome (TAC) Library

It's basal work to construct larger fragment genomic library for genomic research, gene separation and fast using, especially for larger genomic biology. TAC vectors have the P1-plasmid and Ri-plasmid origin of replication, so can shuttle replicate in E.coli and agrobacterium with single copy. Therefore, it can take the gene fuction-complement experiment directly with the agrobacterium -mediated transformation system in case we get the aim clones, so it's not necessary to subclone and it can accelerate the work course.High quality vector DNA and larger fragment DNA are two basal elements for genomic library construction. So far, most papers emphasize particularly on the construction and utilizing of library and the gain of larger fragments DNA, and they take little attention on the research of preparation about vector DNA. In this paper, we did a series of experiments to get high quality vector DNA for construction of TAC library with pYLTAC17 and pYLTAC747H vectors, the contents included abstraction and purification and detecting of plasmids, choosing of complete digested condition with different units HindIII, detecting complete dephosphorylation effect by self-ligated vector, detecting the ligating capability of vector with lambda DNA/Hind III and analysis of transformation colonies. The main results obtained were as follow:The concentration of pYLTAC17 abstracted and purificated by QIAprep Spin Miniprep Kit was 300ng/ul, and pYLTAC747H was 400ng/ul. E.coli ElectroMaxTM DH10BTM competent cells electroporated with the vectors after culturing in 1.0 ml SOC solution , were plated on LB+Kan 25mg.L^-1 (+5 % sucrose) culture medium, the proportion of clonies on culture medium between with and no sucrose was accounted that pYLTAC17 was 1: 28885.7, pYLTAC747H was 1: 119600, they were both far less than 1/5000, so the sacB gene in both vectors were normal and could continue latter experiment. The electrophoresis result of the vectors digested with 1U, 2U,3U,5U,7U Hindlll showed the optimal Hindlll digesting condition was 3U/ug liner vector DNA, 37℃, digesting 30min. The electrophoresis and electroporation competent cells results of the vectors dephosphorylated with 0.5MBU and 1MBU HK showed the optimal HK dephosphorylating condition was IMBU/ug liner vector DNA, 30℃, dephosphorylating 1h. E.coli ElectroMaxTM DH10BTM competent cells electroporated with the ligated DNA between...