BIOL 5060 Lecture Notes - Lecture 11: Sticky And Blunt Ends, Thermal Cycler, Alanine
Document Summary
Mutation may up-regulate/ down-regulate activity or maybe even inactivate it. We are concerned with regulatory sites especially phosphorylation sites= most common regulatory site to rapidly change the function of a protein. Ser and thr= most commonly phosphorylated sites. Changed to 1) alanine non phosphorylat-able version of the protein: 2) aspartic acid or glutamic acid phosmomimetic (mimics phosphorylated form, 1) find your codon of interest and make 2 oligonucleotides of interest*** Remember this: 2 olligonucleotides represent the two strands but with the alteration in the center of the oligos. Altered codon is in the middle; roughly 14-17 nt to either side. Design oligos and use them to replicate plasmid in vitro (like pcr in that it involves heating up the. Dna to melt it apart, cooling it to allow it to anneal to the new oligos and synthesizing the new strand) There is no ligase so, there is a niche (its not a circle)
