Making LB Protocol

Making 1L LB! Liquid or with agar

Need:

At least 1L graduated cylinder

1L Pyrex bottle

Stir Bar

4 250 mL Pryex bottles to distribute LB into

10g Tryptone

5g Yeast extract

5g NaCl

2g MgSO

17.5g Bacto Agar if making plates

1mL Ampicillan per liter LB

Plates

1.    Measure 1L H20 – use milli-Q water. Pour into 1L Pyrex Bottle.

2.    Add stir bar, place on stir plate and set to high

3.    Add 10g BactoTryptone

4.    Add 5g Bacto yeast extract

5.    Add 5g NaCl

6.    Add 2g MgSO4

7.    If making LB plates, add 17.5g Bacto Agar

8.    Let stir until fully dissolved

9.    Check pH, add NaOH until it is at 7.5

10. If making plates, leave in 1L bottle. If NOT making plates, split 1L into smaller 250mL bottles.

11. Autoclave LB.

12. ''If making plates, wait for LB to cool down to the point where you can touch the bottle without any problems. Work on plating inside the sterile hood (make sure to clean hood with bathroom cleaner & spray down w/ 70% EtOH3) ''

13. ''Add 1mL ampicillian/1L LB. Shake vigorously, then wait until bubbles are gone before pouring. ''

13. Label plates.

14. ''Pour LB w/ agar + ampicillan into each plate. Fill each plate about halfway. ''

15. ''Cover stacks of plates with plastic wrap as they solidify. ''

16. Once solid, bag plates and store them in the deli case.