Standard Ligation Reaction

This protocol describes a standard ligation reaction for cloning a PCR fragment using Promega’s pGEM-T Easy Vector system.



5μl       2X Ligation Buffer

.2-.5μl Destination Vector

.5μl      Ligase

Volume to 10μl with water


Incubate at room temperature for 1 hour or overnight at 4°C


Thaw competent cells (TOP10 cells from Invitrogen) on ice (be very careful with these cells!)

Add 3μl of the ligation reaction to the competent cells and gently mix

Incubate on ice for 30 minutes

Heat shock the cells by incubating at 42°C for 45-50 seconds

Immediately place tube on ice for 2 minutes

Add 250μl of pre-warmed S.O.C

Shake at 37°C for 1 hour

Plate out 50μl and 100μl on appropriate plates and incubate overnight at 37°C