November 14, 2021

1. Label 0.6 mL tubes (three different concentrations + positive control + negative control) and prepare an ice bath to incubate in

• Concentrations of KCNJ5_FLAG_pcDNA3.1: 0.1 ng/µL , 1 ng/µL , and 10 ng/µL

2. Put 5 µL of circularized DNA product (of differing concentrations) in their respective tubes and incubate on ice for 10 minutes

3. Take the ice bath to the -80ºC freezer

4. Get a tube of aliquoted JM109 competent cells from the JM109 box (with red tape!)

• Place the tube on ice to let the cells defrost

• 40 µL of cells present in the tube

5. Turn on the water bath while waiting and set it to ~42ºC

• Check on the temperature regularly and adjust temperature dial accordingly

6. Add 10 µL of cells from JM109 to each of the tubes

7. Incubate on ice for 30 minutes

8. Label four 2 mL tubes

9. Add 450 µL SOC media from Fridge B to the 2 mL tubes and place it in the incubator

• Turn on the small incubator/shaker (SHAKIRA SHAKIRAAA) in the equipment room and turn to 37ºC

10. Heat shock the cells in 42ºC water bath for 40 seconds

• Put them on ice for 2 minutes

11. Transfer cells to their corresponding tube with SOC media

12. Shake in the incubator (SHAKIRA SHAKIRAAA) at 170 rpm and 37ºC for 1 hour

• Put the culture plates from Fridge B in the large incubator to dry

13. After incubation is complete, get cells and culture plates

14. Label the plates with the name of the DNA, initials, the date, and the concentration

• KCNJ5_FLAG_pcDNA 3.1 MN AJH 11/14/21 [Concentration]

15. Obtain a 100 mL beaker and fill to ~20 mL with 95% ethanol from Fridge A

16. Ignite the bunsen burner

17. Place the culture plate on a rotating stand

• Pipette (~500 µL) all of the bacterial culture into the plate WITHOUT TOUCHING PLATE

• Dip the metal inoculation loop in ethanol and sterilize in flame

• Let loop cool (~10 seconds)

• Spread bacteria evenly on the plate

• Put the lid on and remove from the stand (ensure that the agar side is down!)

18. Repeat for all plates

• Negative control: ~500 µL water

19. Put the plates in the large incubator at 37ºC to incubate overnight

20. Take pictures of plates in the morning

Results: