IGEM:Virginia 2012/Protocols/LB Broth or Agar Preparation

Overview

Below is the procedure for preparing LB agar plates and LB liquid media.

Materials

• Lennox LB Broth
• Difco agar
• Distilled water
• 2 L bottle or flask
• Foil
• Autoclave tape
• Plastic tray
• Antibiotics
• Petri dishes

Procedure

1. For 1 L of media, measure out 20 g Lennox LB broth. If you're preparing plates, also measure out 15 g Difco agar.
2. Measure 1 L distilled water
3. Add half the water and a stirring magnet to a 2 L bottle or flask
4. Place bottle on stirrer
5. Add reagents from step 1 and let mix
6. Add the remaining water
7. Cover the bottle with foil and a piece of autoclave tape
8. Place bottles in a plastic tray; fill tray with about 1 inch of water
9. Place tray in the autoclave; record date and start time on the sign-in sheet; select cycle (“large liquid”) and start autoclave
10. Remove tray from autoclave
11. Let agar cool to ~55°C (you should be able to touch the jar for at least five seconds)
12. Add sterilized antibiotics, such as chloramphenicol (1 ml, 25 mg/ml) for bronchiseptica. If you prepare drug solutions from scratch, remember to filter sterilize.
13. Remove sterile Petri dishes from plastic bag (save the bag for storage).
14. Pour a thin layer of mixture (~20mL) into each plate, being careful to not lift the cover off excessively (you should be able to just open up enough to pour. If necessary, swirl plate to distribute agar on bottom completely.
15. Let each plate cool until it is solid (~20 minutes), then flip so as to avoid condensation on the agar. Let plates dry overnight (or longer) on bench.
16. Store plates in plastic bags in cold room with: “VGEM”, name, date, and contents (such as type of media and drugs).

Notes

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