NanoBio: LB plates

LB media

• For 1L of media, mix in a 2 L plastic bucket:
  • 20 g LB (Sigma-Aldrich, catalog #)
  • distilled water to 1 L

• Aliquot the liquid media in 500 mL bottles. Do not include a stirrer bar in the bottle. Tighten the cap such that the lid is loose, but cannot be pulled off.
• Autoclave for 15 min at 121C (250F). In 5218, this is Program 09.
• Once the media has cooled to room temperature, tighten the caps.

LB plates

• For ~2 sleeves of plates, mix in a 2 L flask:
  • 20 g LB (Sigma-Aldrich, catalog #)
  • 1 L distilled water
• Dissolve completely.
• Add & mix:
  • 20 g Bacto-agar (BD Biosciences, catalog #)

• Autoclave in 500 mL bottles for 15 min at 121C (250F). In 5218, this is Program 09.
• Allow to cool to ~50C. Add and mix antibiotics as appropriate (refer to working antibiotic concentrations).
  • Note: It is important to cool the media sufficiently such that it is safe to handle and that the antibiotics will not thermally degrade. However, it is also important not to cool so much that the agar solidifies before the plates are poured.
• Pour or pipet ~20 mL of warm media into each plate. If using antibiotics, pouring can be done on the benchtop. If no antibiotics are used, then pour the plates in the Biosafety cabinet.
• After the agar has solidied, ~30 min, invert the plates (agar side up) and allow to dry on the benchtop for ~1-2 days.
• Use colored markers to label the plates.
  • LB Agar = red
  • LB-Amp = blue
  • LB-Chl = red & blue
  • LB-Kan = red & black
  • LB-Tc = red & green

CAjoF 14:41, 21 December 2007 (CST)

In a rush?

If you are in a rush to get a certain antibiotic or combination of antibiotics on an LB plate, here is a simple protocol if you're in a rush. In this example, I needed Kan+Chl plates and had Kan only plates available.

1. Acquire pre-made Kan+LB plates and a stock solution of Chl.
2. Dilute the stock solution of Chl such that you will adding 100μL will dispense the appropriate amount of antibiotic to the gel.
   • Standard gels are 10mL of LB.
   • Our lab uses 30μg/mL Chl plates. See suggested antibiotic concentrations
   • For these conditions, dilute to 3mg/mL Chl.
3. Add 100μL of the diluted antibiotic to the gel and spread with glass beads.
   • 100μL of 3mg/mL Chl in a 10mL plate becomes 30μg/mL.
4. Allow the antibiotic to dry onto the plate at room temperature.
5. Warm the plates at 37°C for ~10 minutes before plating out transformants.

Heather M. Jensen 11 June 2008

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