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Restriction Enzymes • Restriction enzymes cut DNA from any source into fragments • Sticky ends can pair up with complementary DNA with the help of ligase producing recombinant DNA • Example: EcoRI, HindII 2. Must Be Able To Insert Gene Of Interest (GOI) Into A Vector • Vector: plasmid or virus – Plasmid: small circular double stranded DNA – Carries only a few genes – Marker:antibiotic resistance PCR—Polymerase Chain Reaction • • • Denaturation at 94º C DNA disassociates to SS Add annealing primers at 54 º C :enables polymerase to attach Extensions at ~72 º C :taq polymerase & lots of nucleotides PCR • Steps 1-3 are repeated many times • A single fragment of DNA produces – > 1 million copies! – A few hours = 100 billion copies! • Tiny speck of blood and amplify it for crime lab