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From 2010.igem.org

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Revision as of 17:27, 14 June 2010

Contents 1 Notes 2 Materials 3 Method 3.1 Mixing and Sterilizing Media 3.2 Pouring Plates

Notes

• (Optional) pre-heat a H2O bath to 55 deg C.
• If you don't know how to use the autoclave, ask for a demo.

Materials

For 1 L of Media:

• 10 g of Agar (for 1% Agar plates (w/v))
• media components specific to media of interest
• 1 L ddH2O
• Sterile antibiotic stock solution (if required)

Method

Mixing and Sterilizing Media

1. Add dry materials and H2O to a flask sufficiently large to minimize boil-over. (ex.2 L flask for 1 L of media.)
2. (optional, really unnecessary) add a magnetic stir bar and stir the media.
3. Cover the top of the flask LOOSELY with aluminum foil.
4. Autoclave the media on the Fluid Cycle, 30 min
5. Remove media to 55 deg. H2O bath or room temp to cool.
6. Once media has cooled to ~55 deg. C, add antibiotics, if required.

Pouring Plates

1. Obtain 2 sleeves of empty plates. 1 L of media will make about 40 plates.
2. Using sterile technique, pour the media 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 rules:

  Red stripe- AMP
  Blue stripe- KAN
  Green stripe- CHLOR

1. Store the labeled bags of plates in the cold room.

Adapted from the openwetware protocol