Haynes:LB liquid media

LB Liquid Media

by Karmella Haynes, 2012

MATERIALS

• Personal Protective Equipment (PPE) - lab coat, disposable gloves
• OPTION 1
  • Caesin Tryptone
  • Yeast Extract
  • NaCl
• OPTION 2
  • Acros LB Broth, Lennox (BP9722-500)
• 1N NaOH

• One clean and sterile 1000 mL graduated cylinder
• Five clean and sterile 250 mL glass bottles with lids

PROCEDURE

Make LB liquid media - these instructions are for 1 liter

1. Put on your PPE.
2. Fill a 1000 mL graduated cylinder with about 500 mL of deionized water (from the sink)
3. OPTION 1
   1. Weigh out 10 g of Caesin Tryptone on the balance.
   2. Add the Caesin Tryptone to the water in the cylinder.
   3. Weigh out 5 g of Yeast Extract.
   4. Add the Yeast Extract to the mixture in the cylinder.
   5. Weigh out 5 g of NaCl.
   6. Add the NaCl to the mixture in the cylinder.
4. OPTION 2
   1. Weigh out 25 g of Acros LB Broth, Lennox granules.
   2. Add the LB Broth to the mixture in the cylinder.
5. Use a 1000 μL micropipette to add 1000 μL 1N NaOH to the mixture.
6. Swirl the mixture for a few seconds.
7. Fill the cylinder up to 1000 mL with deionized water (from the sink).
8. Get five clean, sterile 250 mL glass bottles with lids from the shelf. Remove the caps and set them aside.
9. Carefully cover the cylinder with a large square of parafilm. Make sure the seal is tight.
10. Cover the opening with your gloved hand and invert the cylinder several times to mix the solution.
11. Quickly and carefully fill one flask up to the 200 mL mark. Cover the cylinder again and invert to mix.
12. Quickly and carefully fill a second flask up to the 200 mL mark. Cover the cylinder again and invert to mix.
13. Repeat until you have filled five flasks to the 200 mL marks.

Sterilize the LB liquid media

1. OPTION 1 - Microwave
   1. Replace the caps with Kimwipe-covered black foam plugs.
   2. Microwave the flasks for 5 minutes. Use a rubber mitt to swirl the flasks (to remove trapped air).

1. OPTION 2 - Autoclave
   1. Loosely put the caps on the bottles. Put a small piece of autoclave tape that is stuck to both the lid and the bottle.
   2. Put the flasks in an autoclave tray. Fill the bottom of the tray about 1 inch deep with tap water (this trick prevents boil-over).
   3. Take the tray with flasks up to the 3rd floor autoclave.
   4. Choose the "Liquid 15" program/cycle. Put the tray with flasks into the autoclave, close the door, and hit "start".