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