IGEM:Brown/2007/Lab Protocols/Pouring Plates 1

Preparation of Agar Plates

1. Agar Recipes: 10 g of NaCl 10 g of Tryptone 5 g of Yeast Extract 20 g of Agar Adjust pH to 7,0 (with 5 N NaOH if necessary) Add deionized Water to a final Volume of 1 Liter.

2. Autoclave (121°C program for solutions, put autoclave-temperature-sensor into water!) 3. Let it cool down in a water bath (adjusted to 55°C)! 4. Add Ampicillin-Na or Kanamycin stock solution to an end concentration of 100 µg/ml 5. Stir or shake accurately to get a homogenous antibiotics distribution! 6. Pour about 25-30 ml in a 10 cm dish 7. Let agar solidify at room temperature, then put to 4°C

Stability of Agar plates: Of course stability problem is the same for medium and plates! Always add antibiotics to LB-Medium freshly and short before use! It is often said, that Ampicillin plates are stable for 4 weeks at 4°C. In fact already after 1 week you can observe reduced selectivity! Therefore I always plate out 25 µl of antibiotic-stock together with bacteria! Picking colonies without wanted content is very annoying. CITATION:Preparing Antibiotics Stock Solution and Ampicillin Agar Plates