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...)
Current revision (17:24, 10 October 2013) (view source)
(LB Liquid Media)
 
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=LB Liquid Media=
 
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by Karmella Haynes, 2012<br><br>
 
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MATERIALS
 
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* Personal Protective Equipment (PPE) - lab coat, disposable gloves
 
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* LB Broth powder (Sigma L3022)
 
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* 1N NaOH
 
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* Two clean and sterile 1000 mL glass flasks
 
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PROCEDURE
 
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'''Make LB''' - these instructions are for 1 liter
 
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# Put on your PPE.
 
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# Fill a 1000 mL graduate cylinder with deionized water (from the sink)
 
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# Weigh out '''20 g of LB Broth powder''' on the balance.
 
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# Add the LB powder to the water in the cylinder.
 
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# Weigh out '''15 g of Bacteriological Agar'''.
 
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# Add the Bacteriological Agar to the mixture in the cylinder.
 
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# Use a 1000 μL micropipette to add '''1000 μL 1N NaOH''' to the mixture.
 
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# Swirl the mixture for a few seconds.
 
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# Fill the cylinder up to 1000 mL with deionized water (from the sink).
 
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# Get '''two clean, sterile 1000 mL glass flasks''' from the shelf. Remove the foil caps and set them aside
 
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# Carefully cover the cylinder with a large square of parafilm. Make sure the seal is tight.
 
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# Cover the opening with your gloved hand and invert the cylinder several times to mix the solution.
 
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# Quickly and carefully fill each flask up to the 300 mL mark. Cover the cylinder again and invert to mix.
 
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# Quickly and carefully fill each flask up to the 400 mL mark. Swirl the cylinder to mix.
 
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# Quickly and carefully fill each flask up to the 500 mL mark.
 
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'''Melt the LB Agar'''
 
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# OPTION 1 - Microwave
 
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## Replace the foil caps with Kimwipe-covered black foam plugs.
 
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## Carry the flasks (in a tray) to the large white microwave (Wang Lab).
 
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## Microwave the flasks for 5 minutes. Use a rubber mitt to swirl the flasks (to remove trapped air).
 
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## Microwave the flasks for another 5 minutes. Use a rubber mitt to swirl the flasks (to remove trapped air).
 
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## Microwave the flasks for10 minutes. Use a rubber mitt to put the flasks back into the tray and bring them back to your bench
 
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# OPTION 2 - Autoclave
 
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## Put the foil caps on the flasks.
 
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## 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).
 
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## Take the tray with flasks up to the 3rd floor autoclave.
 
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## Choose the "Liquid 15" program/cycle. Put the tray with flasks into the autoclave, close the door, and hit "start".
 
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