"
Team:Valencia/LB Agar
From 2012.igem.org
LB Agar Plates
- Weigh 35g of LB-Agar powder mix per liter of media desired. One liter makes 40-50 plates. Ensure that that the mixture volume does not exceed half of the volume of the flask/contained used, as it will boil over in the autoclave.
- Dissolve LB-Agar, using water from one of the wall mounted Nanopure filters. Add a stir bar and use a magnetic stirrer to facilitate mixing.
- Cover the flask with aluminum foil, and secure the foil with autoclave tape. The foil should be somewhat loose (to avoid building pressure in the flask while sterilizing and blowing the foil off), but not so loose that lots of liquid can escape.
- Put the flask in a plastic autoclave tray, load into the autoclave, and sterilize using the 20 minute liquid program.
- Once the autoclave finishes venting (which can take twice as long as the sterilization proper), check that the foil covering is still in place. If it is not, the media is contaminated! Unload using the insulated oven gloves.
- Allow the media to cool until it can be handled without the oven mits. The cold room can be used to speed this up. Alternatively, if a large batch of media is prepared flasks may be kept hot in the prep lab water bath, to avoid all of them cooling at once. Agar polymerization cannot be reversed once it starts, but media can be kept from solidifying by keeping it hot.
- Once media is cool, add other desired ingredients. Use the magnetic stirrer to mix, but do NOT add a stir bar now, or the media will be contaminated. (If one wasn't added before, you must do without.) Common additions include:
- Ampicillin (stock 100mg/ml, final 100µg/ml)
- Kanamycin (stock 50mg/ml, final 50µg/ml)
- Chloramphenicol (stock 50mg/ml, final 10µg/ml)
- To achieve final concentrations, add 1mL of stock per 1L of media, except for chloramphenicol, where 0.6mL per 1L of media is added instead.
- Pour directly from the flask into sterile petri plates. Use a quick pass with a bunsen burner flame to eliminate any bubbles that form during pouring. Do not subject the plate to continuous heat or the plate will melt, and the heat sensitive ingredients added in the previous step will be destroyed. Bubbles can allow cells to access nutrients without being exposed to the plate's antibiotic, and should be blown out immediately before the gel can set. It's a good idea for one person to pour while another flames bubbles.
- Allow the plates to stand right side up overnight, or until the gel sets if they are needed sooner. Plates should be stored upside down to keep condensation from falling on the media. Store petri plates in the plastic bags they ship in, in the 4 degree cold room.
