E116C/R104C/E1371Q

1. Add 5 µL of circularized DNA product (1-50 ng of plasmid DNA) to a previously labeled 0.6 mL tube. Incubate on ice for 10 minutes.

2. Take the cooler with ice and go to the -80°C freezer. Obtain a tube of JM109 competent cells from the JM109 box (red tape). Place the tube on ice and let the cells defrost. While waiting, turn on the water bath and set at 42°C. Periodically check the water bath temperature and adjust the temperature dial accordingly.

3. Add 10uL of cells (if not a PCR product) 40 µL of cells (if a PCR product) to the tubes containing circularized DNA and incubate on ice for 30 minutes. Record the total volume of cells removed from the tube. Subtract that volume from the volume written on the label. Replace the old label with a new one and write the new volume of cells in the tube. Replace the cells in the -80°C freezer.

4. Add 450 µL SOC media (Fridge B) to a previously labeled 2 mL tube and place it in the incubator. Turn on the small incubator/shaker in the equipment room down the hall.

5. Heat shock the cells in the 42°C water bath for 40 seconds and then let them rest on ice for 2 minutes. Transfer the cells to their corresponding tube with SOC media. Shake in the incubator at 170 rpm, 37°C for 1 hour. Place the culture plates (Fridge B) in the large incubator to dry.

6. After the incubation period, retrieve the cells and culture plates. Label the plates with the name of the DNA, your initials, and the date. Obtain a 100 mL beaker fill it about halfway with 95% ethanol (Fridge A) and ignite the Bunsen burner.

7. Place the culture plate on the rotating stand. Pipette all of the bacterial culture into the plate. Dip the metal spreader in the ethanol. Pass the spreader through the flame. Allow the flame to die and let the spreader cool for about 10 seconds. Spread the bacteria evenly on the plate. Replace the lid and remove the plate from the stand ensuring the agar side is down.

8. Place the plates in the large incubator at 37°C and incubate overnight (agar side down).