BIOL 5060 Lecture Notes - Lecture 5: Base Pair, Adenine, Polyethylene Glycol
Document Summary
Preparation of dna= initial step to making recombinant dna genes/ molecules. Restriction enzymes and digests: sau3ai- |gatc, bamhi- g|gatcc. **both leave the same sticky ends= complimentary ends compatible for ligation: both leave a gatc overhang, a|gatct- bglii, t|gatca- bcli. After cutting, you may inactivate enzyme before ligation (if recreating the cut site) Ways of inactivating: heat killing, phenol-chcl3 extract to remove protein, spin columns can remove protein, run dna on gel and elute band of interest, least favorite because may result in a loss of material. Ligation: most common ligase= t4 dna ligase, works for both blunt and sticky ends, e. coli dna ligase- only for sticky ends, taq ligase- thermostabile ligase- not used in plasmid constructions, used at high temperature. ** used to create sticky ends on blunt dna. Polynucleotide kinase- adds 5" phosphates to dna; add labeled phosphate to dna from gamma-32 p-atp.
