BCMB20005 Lecture Notes - Lecture 2: Complementary Dna, Dna Extraction, Isopropyl Alcohol
2 Aug 2018
School
Department
Course
Professor
Document Summary
Step 2. a)obtain dna fragment of interest: use restriction enzymes to cut" dna at specific sites. Screen for fragment of interest** b)choose cloning vector. Usually a plasmid (small circular dna molecule, independently replicating) Step 4, insert dna fragment into cloning plasmid: use dna ligase. Isolating dna (step 1) using alkaline lysis method: source dna (e. g. e. coli bacteria transfected with puc19) i. ii. iii. Discard supernatant (media/serum) - contain waste from cell and nutrient medium that it was grown in. Resuspend cell in tris/edta solution - pipette up and down. Tris = buffer, maintain ph at 8. Edta = inhibit dna that might be present when it burst open and might release. It does this by chelating to the divalent cations that are acquired by the dnas for their enzyme activities. Inhibit and prevent them from chopping up any dna: lyse cells to release contents (dna, rna and proteins) Chemical lysis (e. g. add alkaline + detergent)
