BY 108 Chapter Notes - Chapter 16: Restriction Enzyme, Molecular Cloning, Southern Blot
Document Summary
Dna cleavage (stage 1) - restriction endonuclease cleaves dna into fragments. Gel electrophoresis - procedure that separates fragments based on size recombinant dna production (stage 2) - dna fragments inserted into vectors. Vectors cleaved w/ same restriction endonuclease as dna cloning (stage 3) - more recombinant dna created. Each reproduced cell contains recombinant dna screening (stage 4) - most challenging part of any genetics experiment. Gets rid of cells w/o any vectors, vectors w/o original dna. Uses vector w/ gene for antibiotic resistance. Based on presence/absence of a certain phenotype. Hybridization - uses complementary nucleic acid (probe) to find particular fragment. Solution added to denature dna, allowing radioactive probe to attach polymerase chain reaction (pcr) - uses dna polymerase to mass produce gene sequences. Denaturation (step 1) - excess of primer mixed w/ dna fragment. Dna strand heated to 98 c >> strands dissociate. Annealing of primers (step 2) - dna solution allowed to cool to 60 c.
