Cloning into Plasmids.ppt

Cloning into Plasmids Restriction Fragment Cloning PCR Cloning by the Topo TA? Method Cloning Vectors The molecular analysis of DNA has been made possible by the cloning of DNA. The two molecules that are required for cloning are the DNA to be cloned and a cloning vector. Cloning vector - a DNA molecule that carries foreign DNA into a host cell, replicates inside a bacterial (or yeast) cell and produces many copies of itself and the foreign DNA Three features of all cloning vectors sequences that permit the propagation of itself in bacteria (or in yeast for YACs) a cloning site to insert foreign DNA; the most versatile vectors contain a site that can be cut by many restriction enzymes a method of selecting for bacteria (or yeast for YACs) containing a vector with foreign DNA; uually accomplished by selectable markers for drug resistance Types of Cloning Vectors Plasmid - an extrachromosomal circular DNA molecule that autonomously replicates inside the bacterial cell; cloning limit: 100 to 10,000 base pairs or 0.1-10 kilobases (kb) Phage - derivatives of bacteriophage lambda; linear DNA molecules, whose region can be replaced with foreign DNA without disrupting its life cycle; cloning limit: 8-20 kb Cosmids - an extrachromosomal circular DNA molecule that combines features of plasmids and phage; cloning limit - 35-50 kb Bacterial Artificial Chromosomes (BAC) - based on bacterial mini-F plasmids. cloning limit: 75-300 kb Yeast Artificial Chromosomes (YAC) - an artificial chromosome that contains telomeres, origin of replication, a yeast centromere, and a selectable marker for identification in yeast cells; cloning limit: 100-1000 kb General Steps of Cloning with Any Vector prepare the vector and DNA to be cloned by digestion with restriction enzymes to generate complementary ends (exception Topo cloning see later slides) ligate the foreign DNA into the vector with the enzyme DNA ligase introduce the DNA into bacterial cells (or yeast cells for YACs)