|
|
| Line 1: |
Line 1: |
| =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".
| |
|
| |
|
| |
|
| |
|
| |
| </div>
| |
