Identification of a Plasmid: Transformation

Transfer 1mL E. Coli to each of two 1.5mL Eppendorf tube

Centrifuge at full speed for 0h 0m 30s, then remove the supernatant

Add 450µL of TFB1 to each tube, then resuspend gently by pipetting up and down (do not vortex)

Keep everything On ice

Incubate on ice for 0h 10m 0s

Pellet the bacteria by centrifugation at full speed for 0h 0m 30s

Remove the supernatant

Resuspend the pellets in 100µL of TFB2

Incubate on On icefor 0h 10m 0s

Add 100ng of plasmid DNA to the competent cells, and mix gently

Heat shock the cells by transferring directly from the ice to the 37°C hot block for 0h 5m 0s

Then incubate on ice for 0h 5m 0s, and add 1mL LB broth to each tube

Incubate again at 37°C, for 0h 30m 0s, and allow the cells to recover

Pellet the bacteria again by centrifuging at full speed for 0h 0m 30s

Discard the supernatant

Resuspend the pellet in 400µL of LB broth

Spread 100µL of cells per plate, onto the appropriate antibiotic plates

Incubate the plates 0h 0m 30s at 37°C