Bio 99 Midterm 1 Lec: 9

• Express proteins in different organisms
• ex.) Bacterial Multi-Drug Resistance Pump ----> human cells
• Express modified versions of proteins
• Purify proteins 
• Create DNA libraries

• Allow us to genetically manipulate multicellular organisms at a single cell level
• Allow us to visualize processes that are difficult to observe even with the most advanced microscopes

B.

restriction enzyme digestion (put on plate and choose which one you want)

copied DNA

• 1. Blunt ligate the duplex cDNA into vector
• 2.
• a. Blunt ligate RE sites to the ends of the DNA
• b. Clone the cDNA into vector with the same “sticky ends”

• •As source material to clone a gene
• •Identification of a mutated gene (functional complementation)
• •Genome sequencing

• •Classical/Reverse genetics
• –Start with wild type organism ----> mutate to obtain organisms with a particular phenotype
• •Once you have your phenotype, how can we determine which gene was mutated?

used for selection against certain cells unable to survive in this selective temperature. For example, in case of temperature-sensitive mutants permissive temperature will be 230C (all of the cells will form colonies), and 360C will be selective temperature that will select against temperature-sensitive mutants(they will be unable to form colonies).

• •Sanger sequencing
• – ddNTPs-dideoxy chain termination method

• Terminator Nucleotides
• Both fluorescent dye and 3’ OH blocking molecule are removed before next nucleotide addition