Haynes:LB liquid media

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(New page: =LB Liquid Media= by Karmella Haynes, 2012<br><br> MATERIALS * Personal Protective Equipment (PPE) - lab coat, disposable gloves * LB Broth powder (Sigma L3022) * 1N NaOH * Two clean and...)
(LB Liquid Media)
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MATERIALS
MATERIALS
* Personal Protective Equipment (PPE) - lab coat, disposable gloves
* Personal Protective Equipment (PPE) - lab coat, disposable gloves
-
* LB Broth powder (Sigma L3022)
+
* Caesin Tryptone
-
 
+
* Yeast Extract
 +
* NaCl
* 1N NaOH
* 1N NaOH
-
* Two clean and sterile 1000 mL glass flasks
+
* 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
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'''Make LB''' - these instructions are for 1 liter
'''Make LB''' - these instructions are for 1 liter
# Put on your PPE.
# Put on your PPE.
-
# Fill a 1000 mL graduate cylinder with deionized water (from the sink)
+
# Fill a 1000 mL graduated cylinder with about 500 mL of deionized water (from the sink)
-
# Weigh out '''20 g of LB Broth powder''' on the balance.  
+
# OPTION 1
-
# Add the LB powder to the water in the cylinder.
+
## Weigh out '''10 g of Caesin Tryptone''' on the balance.  
-
# Weigh out '''15 g of Bacteriological Agar'''.
+
## Add the Caesin Tryptone to the water in the cylinder.
-
# Add the Bacteriological Agar to the mixture 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.
# Use a 1000 μL micropipette to add '''1000 μL 1N NaOH''' to the mixture.
# 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 '''two clean, sterile 1000 mL glass flasks''' from the shelf. Remove the foil caps and set them aside
+
# 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.
# 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 each flask up to the 300 mL mark. Cover the cylinder again and invert to mix.
+
# Quickly and carefully fill one flask up to the 200 mL mark. Cover the cylinder again and invert to mix.
-
# Quickly and carefully fill each flask up to the 400 mL mark. Swirl the cylinder 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 each flask up to the 500 mL mark.
+
# Repeat until you have filled five flasks to the 200 mL marks.
-
'''Melt the LB Agar'''
+
'''Sterilize the LB liquid media'''
# OPTION 1 - Microwave
# OPTION 1 - Microwave
-
## Replace the foil caps with Kimwipe-covered black foam plugs.
+
## Replace the caps with Kimwipe-covered black foam plugs.  
-
## Carry the flasks (in a tray) to the large white microwave (Wang Lab).  
+
## Microwave the flasks for 5 minutes. Use a rubber mitt to swirl the flasks (to remove trapped air).  
## Microwave the flasks for 5 minutes. Use a rubber mitt to swirl the flasks (to remove trapped air).  
-
## Microwave the flasks for another 5 minutes. Use a rubber mitt to swirl the flasks (to remove trapped air).
+
 
-
## Microwave the flasks for10 minutes. Use a rubber mitt to put the flasks back into the tray and bring them back to your bench
+
# OPTION 2 - Autoclave
# OPTION 2 - Autoclave
-
## Put the foil caps on the flasks.  
+
## 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).
## 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.  
## Take the tray with flasks up to the 3rd floor autoclave.  

Revision as of 12:58, 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 - 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".



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