Haynes:LB liquid media: Difference between revisions
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PROCEDURE | PROCEDURE | ||
'''Make LB''' - these instructions are for 1 liter | '''Make LB liquid media''' - these instructions are for 1 liter | ||
# Put on your PPE. | # Put on your PPE. | ||
# Fill a 1000 mL graduated cylinder with about 500 mL of deionized water (from the sink) | # Fill a 1000 mL graduated cylinder with about 500 mL of deionized water (from the sink) |
Revision as of 11:00, 11 October 2013
LB Liquid Media
by Karmella Haynes, 2012
MATERIALS
- Personal Protective Equipment (PPE) - lab coat, disposable gloves
- Caesin Tryptone
- Yeast Extract
- NaCl
- 1N NaOH
- ALTERNATIVE: Acros LB Broth, Lennox (BP9722-500) instead of Caesin Tryptone, Yeast Extract, and NaCl
- 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
- 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, sterile 250 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 flask up to the 200 mL mark. Cover the cylinder again and invert to mix.
- Quickly and carefully fill a second flask up to the 200 mL mark. Cover the cylinder again and invert to mix.
- Repeat until you have filled five flasks to the 200 mL marks.
Sterilize the LB liquid media
- OPTION 1 - Microwave
- Replace the caps with Kimwipe-covered black foam plugs.
- Microwave the flasks for 5 minutes. Use a rubber mitt to swirl the flasks (to remove trapped air).
- OPTION 2 - Autoclave
- Loosely put the caps on the bottles. Put a small piece of autoclave tape that is stuck to both the lid and the bottle.
- 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).
- 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".