Directional cloning of DNA molecules

Create nonidentical, complementary sticky ends (directional cloning). Vector DNA Insert DNA EcoRl BamHl EcoRl BamHl _ Digest with both Digest with EcoRI and BamHl. both EcoRI and BamHI. Remove fragment. Nonidentical , \ sticky ends complementary _ / \ to insert— DNA / ends. \\ 4— .....__..__§. /‘G3' 5'6ATCC\ ,CTTAAS' 3'§_\ Combine fragments. Recombinant vector Unligated vector / \ l/ \ \ l/ \\ / \ I/ \ /GA /‘E3' 5'GATCC\ ,c__TTAAHfi: ,CTTAAS’ 3'35) lnserted DNA can be ligated into l lVector alone cannot vector in only one orientation. lbe religated.