Barboza's:Preparing LB agar plates

Preparing LB Agar plates Submitted by User:Uriel E Barboza Perez

Overview

Preparation of LB agar plates

Materials

For the preparation 500 mL of LB Agar

• LB Agar
• 500 mL Pyrex container
• Sterile Water
• Appropriate Antibiotic *
• 17.5 grams of LB Agar
• Desired amount of Petri dishes **

Procedure

1. Weigh 17.5 grams of LB Agar powder, add into sterilized Pyrex container.

2. Fill with sterile water up to 500 mL mark.

3. Cap loosely and tape with autoclave tape.

4. Autoclave until it reaches 120 °C .

5. Once it reaches 120 °C count 15 min ( try to keep the temp at 120 °C during those 15 minutes) and then turn down the temperature until it reaches 0°C.

6. Take the container out and wait until it is cool enough that it doesn't hurt to touch.

If starting from solid agar in the fridge, heat in microwave (cap unscrewed) 3min on high, 5 min on 30% (till it melts completely) Wait till cool enough so that you can touch it for a minute (60 degree Celsius)

7. Add appropriate volume of Amp or Kan (1000X). For 500 mL of LB Agar, add 500 uL of each antibiotic needed.

8. Swirl to mix, use pipette to transfer 20 mL into each plate or simply pour it carefully in the plate . Try to the formation of bubbles. ***

9. Allow plates to cool and solidify. Mark antibiotic on side of plate.

If LB Agar solidifies before being poured, microwave to liquefy again

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

• place antibiotic if needed.
  • this amount of medium can easily make around 20 petri dishes.
    • if bubbles are made , swirl the plate trying to pop the bubbles. If they don't pop , try to move the bubbles to the sides.

Please sign your name to your note by adding '''*Uriel E Barboza Perez''': to the beginning of your tip.