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Revision as of 19:08, 21 September 2013
LB Agar Plates
by Karmella Haynes, 2012
- Personal Protective Equipment (PPE) - lab coat, disposable gloves
- ~40 clean, sterile petri dishes (pr "plates"), packed in plastic "sleeves" per 1 liter of LB agar
- LB Broth powder (Sigma L3022)
- Bacteriological Agar
- 1N NaOH
- Two clean and sterile 1000 mL glass flasks
- 100 mg/mL Ampicillin from the -20°C freezer (if you are making amp plates)
Make LB Agar - 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".
Set up the empty plates - complete this part while the LB agar is melting in the microwave or autoclave
- Cover the work area with bench paper
- Get a stack of plates and cut open the very top of the sleeve. Do not rip apart the sleeve. You will need this to store the finished plates.
- Take the plates out and stack them in sets of five, lid-side-up.
- If you are making Amp plates, take a tube of 100 mg/mL ampicillin from the -20°C freezer and thaw it in your hand or at room temperature.