Difference between revisions of "Haynes:LBliquidmedia"

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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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=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 sterile 1000 mL glass flasks
 
 
 
 
PROCEDURE
 
 
'''Make LB''' - these instructions are for 1 liter
 
# Put on your PPE.
 
# Fill a 1000 mL graduate cylinder with deionized water (from the sink)
 
# Weigh out '''20 g of LB Broth powder''' on the balance.
 
# Add the LB powder to the water in the cylinder.
 
# Weigh out '''15 g of Bacteriological Agar'''.
 
# Add the Bacteriological Agar 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 '''two clean, sterile 1000 mL glass flasks''' from the shelf. Remove the foil 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 each flask up to the 300 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 each flask up to the 500 mL mark.
 
 
'''Melt the LB Agar'''
 
# OPTION 1 - Microwave
 
## Replace the foil 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 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
 
## Put the foil caps on the flasks.
 
## 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".
 
 
 
 
 
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Latest revision as of 14:24, 10 October 2013