Ligation and Transformation[Cloning]

Ligation

1 Add DNA and Insert to a 1.5mL falcon tube at a 1:3 molar ratio in a total volume of 10uL
2 Add 2x ligation reaction buffer (10uL)
3 Add 1uL of ligase to each reaction and let stand on bench top for 5 minutes

Transformation

1 Add 50uL of cells/ transformation
2 Add 1-2ug of DNA (1 or 1.5uL of ligation reaction) and leave on ice 10min
3 Heat shock 42C for 40s
4 Place tubes on ice for 10min
5 Add 250uL of LB or SOC
6 Place at 37 C, shaking ~230rpm for 30min to 1hour
7 Plate 10uL and 20uL for each reaction on plate w/selection

September '06 tj