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- Melt LB ahead of time because it cools to 55 degrees slowly. A good way to do this is melt it in the morning then leave it in the 60 degree incubator or a 60 degree bath. If in a rush, carefully cool by running cold water over the container.
- LB Agar 1.2% (from the media room)
- Empty plates (above any of the -20 freezers)
- antibiotics (liquid stocks from our -20 freezer)
Preparatation of LB Agar
- Obtain 1.2% agar from the media room (500 ml per bottle).
- Be sure to sign it out
- Melt in microwave:
- use 50% power
- loosen the cap
- monitor as you melt
- takes approx. 10 minutes per bottle.
- If too hot, let the melted solution cool so that it's warm, but not hot, to the touch - you can use the water bath to do this (50-60 degrees); this won't let it resolidify. At room temp. it may take 20 mins for LB to cool sufficiently.
- ONCE IT'S COOL ENOUGH (50-60 degrees) - add antibiotics: final concentration, 50ug/ml AMP; 20ug/ml KAN. (concentrations of liquid stocks: Amp; 50mg/ml. Kan, 10mg/ml.) For 500mL of LB Agar:
- use 500uL Amp
- 1mL Kan.
- Swirl to mix; try not to make many bubbles.
- Obtain a container of empty plates. 1 bottle (500ml) of LB Agar will make about 1 container of plates (20 plates).
- Using sterile technique, (flame the top of the bottle), pour the LB Agar into the plates. How much to pour? Cover the base of the plate, and then just a bit more after that.
- Recap each plate upon pouring. If there are lots of bubbles in your plates (i.e. more than one or two on the edge), you can flame the plate using the small bunsen burner to eliminate bubbles. (See a demo on this).
- Leave plates to dry and cool for a while (overnight even). LABEL the stack of plates to indicate antibiotic!
- Store the plates in their original bags - upside down, so that the gel is hanging downwards (this keeps condensation off the gel).
- Label the bags following taping rules:
- Red tape in back (indicates LB)
- Tape in front indicates antibiotic. (Green: Kan. Yellow: Amp.) (More taping/color rules are on the refrigerator in the second bench room)
- also write name of antibiotic, and concentration, on the front piece of tape.
- Store the labeled bags of plates in the cold room.