LB selection plates

  1. Dissolve 32 g of LB agar powder in 800 mL of sterile/ELGA water.

  2. Add stir bar to the bottle, leave unscrewed, and cover with aluminum foil.

  3. Autoclave on 20 min liquid cycle at 121C.

  4. Wait for the solution to cool to about 45-50 C by putting on stir plate at room temperature.

Tip

If you can keep touching it with bare hands, that indicates the temperature of the LB is around 45-50C and it’s time to add antibiotics.

  1. Add 800 uL of 1000x antibiotics to the autoclaved LB agar-water solution.

  2. Dissolve the antibiotics by stirring with a stir bar.

  3. Before pouring, ensure no agar has solidified in the bottle (as a result of uneven cooling). If this happens, shake manually to re-melt the agar.

  4. Pour ~12 mL of the antibiotic-containing LB to petri dishes using a pipette.

  5. Gently move the plates around to evenly distribute the agar.

Tip

Use a 50 mL serological, fill to 60 mL, and pipette out 12 mL at a time onto sets of 5. Alternatively, you can pour LB by hand if you experience solidification when pipetting.

  1. Incubate the poured plates at RT until the LB agar solidifies (~1 hr minimum-overnight).

  2. After plates solidify, place plates back in the sleeve bag and store at 4C.