Haynes:LB liquid media: Difference between revisions

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* One clean and sterile 1000 mL graduated cylinder
* One clean and sterile 1000 mL graduated cylinder
* Five clean and sterile 250 mL glass bottles with lids
* Five clean and sterile 250 mL glass bottles with lids (square-shaped bottles)




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# Swirl the mixture for a few seconds.
# Swirl the mixture for a few seconds.
# Fill the cylinder up to 1000 mL with deionized water (from the sink).
# 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.
# 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.
# 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.
# 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 one bottle 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.
# 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 flasks to the 200 mL marks.
# 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'''
'''Sterilize the LB liquid media'''
# OPTION 1 - Microwave
# 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.  
## Replace the caps with Kimwipe-covered black foam plugs.
# 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).
## Microwave the flasks for 5 minutes. Use a rubber mitt to swirl the flasks (to remove trapped air).
# Take the tray with flasks up to the 3rd floor autoclave.  
## Microwave the flasks for another 5 minutes. Use a rubber mitt to swirl the flasks. Use a rubber mitt to put the flasks back into the tray and bring them back to your bench.
# 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.
# OPTION 2 - 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.
## Loosely put the caps on the bottles. Put a small piece of autoclave tape that is stuck to both the lid and the bottle.  
# Place the bottles on the appropriate shelf.
## 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".

Latest revision as of 13:51, 6 September 2014

LB Liquid Media

by Rene Davis, 2013

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 (square-shaped bottles)


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, sterilized 200 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 bottle up to the 200 mL mark. Cover the cylinder again and invert to mix.
  12. Quickly and carefully fill a second bottle up to the 200 mL mark. Cover the cylinder again and invert to mix.
  13. Repeat until you have filled five bottles to the 200 mL marks.
  14. Using lab tape, label each bottle with "Liquid LB Lennox," your initials, and the date.

Sterilize the LB liquid media

  1. 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.
  2. 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).
  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".
  5. After about 1 hour, put on a pair of thick orange protective gloves and retrieve the bottles from the autoclave.
  6. 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.
  7. Place the bottles on the appropriate shelf.
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