Haynes:LB liquid media
LB Liquid Media
by Rene Davis, 2013
- Personal Protective Equipment (PPE) - lab coat, disposable gloves
- OPTION 1
- Caesin Tryptone
- Yeast Extract
- 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 (square-shaped bottles)
Make LB liquid media - these instructions are for 1 liter
- Put on your PPE.
- Fill a 1000 mL graduated cylinder with about 500 mL of deionized water (from the sink)
- OPTION 1
- Weigh out 10 g of Caesin Tryptone on the balance.
- Add the Caesin Tryptone to the water in the cylinder.
- Weigh out 5 g of Yeast Extract.
- Add the Yeast Extract to the mixture in the cylinder.
- Weigh out 5 g of NaCl.
- Add the NaCl to the mixture in the cylinder.
- OPTION 2
- Weigh out 25 g of Acros LB Broth, Lennox granules.
- Add the LB Broth to the mixture in the cylinder.
- Use a 1000 μL micropipette to add 1000 μL 1N NaOH to the mixture.
- Swirl the mixture for a few seconds.
- Fill the cylinder up to 1000 mL with deionized water (from the sink).
- Get five clean, sterilized 200 mL glass bottles with lids from the shelf. Remove the caps and set them aside.
- Carefully cover the cylinder with a large square of parafilm. Make sure the seal is tight.
- Cover the opening with your gloved hand and invert the cylinder several times to mix the solution.
- Quickly and carefully fill one bottle up to the 200 mL mark. Cover the cylinder again and invert to mix.
- Quickly and carefully fill a second bottle up to the 200 mL mark. Cover the cylinder again and invert to mix.
- Repeat until you have filled five bottles to the 200 mL marks.
- Using lab tape, label each bottle with "Liquid LB Lennox," your initials, and the date.
Sterilize the LB liquid media
- Loosely screw the caps on the bottles. Apply a small piece of autoclave tape (that has one end folded to make a tab) that is stuck to both the lid and the bottle.
- Put the flasks into an autoclave tray. Fill the bottom of the tray about 1 inch deep with tap water (this trick prevents boil-over).
- Take the tray with flasks up to the 3rd floor autoclave.
- Choose the "Liquid 15" program/cycle. Put the tray with flasks into the autoclave, close the door, and hit "start".
- After about 1 hour, put on a pair of thick orange protective gloves and retrieve the bottles from the autoclave.
- Pull the autoclave tape off the bottle (do not throw the tape away), screw the cap on tightly (but don't over-tighten), and re-apply the autoclave tape.
- Place the bottles on the appropriate shelf.