Pouring LB plates from prepped media-PDF

Notes

• 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, do not attempt to cool the media rapidly by running cold water over the container as this can cause the glass to shatter. Instead, media can be left at room temperature to cool more rapidly.

Materials

• LB Agar 1.2% (from the media room)
• Empty plates (above any of the -20 freezers)
• antibiotics (liquid stocks from our -20 freezer)

Method

Preparation of LB Agar

1. Obtain 1.2% LB agar from the media room (500 ml per bottle).
   • Be sure to sign it out
2. Melt in microwave:
   • loosen the cap
   • use 50% power (enter time, press Power, 5, Start)
   • monitor as you melt
   • takes approx. 10 minutes per bottle.
3. 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.
4. 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:
   • 500μL Amp stock
   • 1mL Kan stock
5. Swirl to mix; try not to make many bubbles.
   • I usually let sit @ 55C for 3-4 min after swirling to let the bubbles go down.

Actual Pouring

1. Obtain a container of empty plates. One bottle (500ml) of LB Agar will make about one container of plates (20 plates).
2. Using sterile technique (flame the top of the bottle)(why? what are you killing here? the plastic sealing ring?), pour the LB Agar into the plates.
   • Cover the base of the plate, and then just a bit more after that.
3. 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). Another way to remove the fine bubbles that may be in your flask before pouring is to mist the inside of the flask with a 75% ethanol spray bottle.
4. Leave plates to dry and cool for a while (overnight even).
   • It is a good idea to label the stack of plates to indicate antibiotic.
5. Store the plates in their original bags – upside down, so that the gel is hanging downwards (this keeps condensation off the gel).
6. 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 68-564)
   • Also write name of antibiotic, and concentration, on the front piece of tape.
7. Store the labeled bags of plates in the cold room.