BIOS 452 Lecture Notes - Lecture 23: Digestion, Hydrophile, Ribozyme
Document Summary
Digestion with bamhi and ecori generates 2 separate dna arms with 1 telomereic end+selectable marker amplify linear genes of interest amplify circular plasmids. Primers containing sequences complementary to target + restriction site sequence to be introduced. Protocol: place in thermocycler, melt dna at 95 degrees, cool to 50-60, primers anneal to template, polymerase extends primers 5"-3", repeat. Simplified: strand separation 98, annealing 65, extension 72. Ligation reaction vector dna(cut and purified) insert dna(pcr product) 3 outcomes of transformation: empty vector- e. coli coloneis will form(xgal is intact blue colonies, vector with insert- white coloneis(xgal is disrupted) Insert self ligation- no colonies because no antibiotics resistance. Separation of dna by electrophoresis: negatively charged dna migrates to the anode in presence of electric field. Poly a tail serves as a universal primer. Reverse transcription results in a dna/rna hybrid where dna strand is complement to mrna mrna-mrna/dna->duplex/cdna lecture 20.
