质粒电泳的三条带2(英文版).docVIP

Method: Plasmid Subcloning May 20 1990 Matthew S. Holt Purpose: The method is used to clone smaller portions of inserts (up to 15 kb in size) which previously have been cloned in YACs phage cosmids or other plasmids. Time required: Plasmid Vector Preparation Restriction digest and Calf Intestinal Phosphatase (CIP) reaction - 4-6 hours or overnight Gel electrophoresis- 4-8 hours or overnight Fragment elution and purification - 4-6 hours Insert Preparation Restriction digest -4-6 hours or overnight Gel electrophoresis- 4-8 hours or overnight Fragment elution and purification - 4-6 hours Ligation Reaction - minimum of 4 hours, best overnight Special Reagents: 10X CIP Buffer 10X Ligation Buffer Preface: Bacterial plasmids are double-stranded circular DNA molecules of 1- 200 kb in size that replicate and are inherited independently of the bacterial chromosome. Different plasmids replicate to different extents in their host some reaching a copy number as high as 700 per cell. They function as accessory genetic units frequently containing genes coding for enzymes that are advantageous to the bacterial host. The most commonly conferred phenotype by plasmids is antibiotic resistance though plasmids can also code for antibiotic production restriction and modification enzymes colicins enterotoxins and enzymes used to degrade complex organic compounds. More than one plasmid can occur in a single cell but since they compete for the same set of replication enzymes one plasmid usually dominates the others. Over the course of a few generations the minority plasmids are completely eliminated and the descendants of the original cell will contain only one of the original plasmids. Over 30 different incompatibility plasmid groups have been identified. Under natural conditions many plasmids are transmitted to a new host by a process known as bacterial conjugation. Newer plasmid vectors however lack the nic/bom site and cannot be conjugated. In the laboratory plasmid DN