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Amgen Lab 2a & 4a Lab 2a: Restriction Enzymes • Restriction enzymes are naturally found in Bacteria & Archaea . • They are thought to provide protection against invading viruses by hydrolyzing “foreign” DNA. • RE cut DNA at specific recognition nucleotide sequences called restriction sites – Called “molecular scissors” Produces “sticky ends” Engineering the Plasmid: ligation of rfp gene into p-ARA Bruce Wallace BamH I sticky end Hind III sticky end Hind III sticky end BamH I sticky end pARA-R construct Bruce Wallace Recombinant plasmid of interest pARA-R 4720 bp rfp 702bp Restriction digest of pARA-R Bruce Wallace Recombinant plasmid of interest pARA-R 4720 bp rfp 702bp Restriction analysis of pARA-R Bruce Wallace Restriction fragments after digest with Hind III and BamH I BamH I Hind III 4018 bp BamH I Hind III 702bp Restriction analysis of pARA-R Prediction for restriction gel M A+ AM A+ A10000 8000 5000 4000 3000 2000 1500 1000 500 A- Lane: Nicked, Multimer, & “Regular” plasmid Ladder A+ A-
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