Rutgers:Transformation

Bacterial transformation(IMPORTANT: keep the cc on ice as much as possible)

• set a heating block at 42°C and fill wells with DW
• defrost a 2 ml tube of SOC medium
• place the required LB plates at 37°C
• set 15 1.5 ml tubes in a recipient full of watery ice
• cut the head of a yellow tip with a razor blade to increase its diameter
• take a tube of XL2-blue competent cells (cc) out of the -80°C freezer, put it immediately in watery ice and wait until it thaws
• gently stir the cc with the pipette tip, slowly resuspend them
• distribute 10 µl of cc into 10 of the 15 tubes
• keep the nulber of tubes needed and put the extra ones at -80°C
• add 0.18 µl of ß-MAE to each tube
• incubate on watery ice for 10 min, gently shaking with a finger tip 2-3 times during the 10 min
• add 1.2 µl of each ligation product into each cc-tubes
• gently mix
• incubate on watery ice for 30 min
• heat shock exactly 30 sec at 42°C in the heating block (no mix, no shake)
• put on ice during 2 min
• spin
• add 125 µl of SOC medium to each tube
• shake the tubes tilted @ 37°C for 1 h at 225 rpm

• during this hour, finish preparing the LB plates (stored @ 37°C):

- prepare the appropriate number of Pasteur pipettes close tip and bend - clean bench using EtOH

• when the incubation is complete, spread 145 µl of SOC-CC onto plate
• let soak 10 min @ 37°C
• incubate inverted @ 37°C overnight
• shift to 4°C for 1 h for color development before screening
• seal the plates with parafilm and store them at 4°C