Biochemistry 2280A Lecture Notes - Chain Termination, Plasmid, Autoradiograph

• School: Western University
• Department: Biochemistry
• Course: Biochemistry 2280A
• Professor: Eric Ball

Document Summary

2", 3" dideoxynucleotides: figure 10. 20, dideoxy ntps act as chain termination, dideoxy lacks 3" oh so it can"t add another nucleotide, stops the chain - chain terminators, key to chain termination sequencing. Materials required to sequence a dna: dna to be sequences (template, oligonucleotide primer that anneals to the template, dna polymerase, dntps (g, a, t, c, ddntps (small amount - 0. 2% of dntp) Plasmid for expressing yfp in e. coli: figure 10. 24, promotor driving your gene; bacterial promotor that you insert before inserting. Yfg: contains strong promotor, ribosome binding site and atg downstream from promotor, right after atg, there is a useful cloning site into which you can insert yfg. Converting a codon for aspartic acid to a codon for alanine: figure 10. 34, anneal/hybridize oligonucleotides to the single stranded plasmid, transform into e. coli, some will have wild type gene, some will have mutant gene (about. 50/50: can generate the mutation within yfg.