Biochemistry 2280A Lecture Notes - Lecture 7: Intron, Chromosome, Genetically Modified Organism

• School: Western University
• Department: Biochemistry
• Course: Biochemistry 2280A
• Professor: Christopher Brandl

Document Summary

Expressing yfp in e. coli: determine which tissues express yfg (hybridization, northern blot) 2: synthesize cdna, pcr yfg from cdna, gel purify pcr product of yfg (gel electrophoresis, restriction cut tfg, ligase, transform into e. coli, verify clones of yfg (restriction mapping, dna sequencing) Will reseal compatible sticky ends; the ends anneal weakly through hybridization. Ligase will recognize the ends, and in the presence of atp will reseal the ends by forming covalent bonds between them. Ligase requires a 5" phosphates to stick the dnas back together. Can also perform the same process with blunt ends, but is more difficult as the sticky ends start off with some attraction to each other already due to weak hybridization. Digest ~100ng of yfg and a plasmid vector with the same restriction enzyme or one that gives compatible overhanging sequences. Most convenient when using the same enzyme.