Take tubes with 50 \u00b5L of TOP10F\u2019 competent cells from -80\u00b0C freezer and place on ice.Add 5 \u00b5L of each ligation reaction directly to competent cells and mix by tapping gently. Do not mix cells by pipetting up and down. Store any remaining ligation at -20\u00b0C.Incubate cells on ice for 30 min. [while waiting, turn on 42\u00b0C water bath]Incubate for exactly 30 sec in 42\u00b0C water bath. Do not mix or shake.Remove vials and place quickly on ice. Store for 2 min on ice.Add 500 \u00b5L of LB medium (pH. 7.5) to each tube.Incubate tubes (taped horizontally to platform) for 60 min at 37\u00b0C and 225 rpm.Plate 550 \u00b5L (variable) of each transformation on LB + AB plates.Incubate overnight at 37\u00b0C, in the dark.