Pelling:Protocols/Bacterial Culture

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Plating Bacteria

  1. From frozen stock (stored in -80°C freezer in 25% glycerol solution).
  2. Scrape surface with a 250μL pipette tip or inoculation loop.
  3. Spread in a line on agar plate.
  4. Use a second pipette tip or loop to generate single colonies (drag the tip lightly in a sine wave pattern, crossing the first line a couple times, repeat in the perpendicular direction).
  5. Seal the plate with parafilm and incubate upside-down overnight at 37°C in the shaker (no shaking).
  6. Plates may be stored in fridge after growth in case of mistakes for up to two weeks.

Growing a 3mL Culture

  1. In 5mL sterile test tubes add 3mL of LB Broth, add appropriate antibiotic (Kanamycin to a final concentration of 50μg/mL or Ampicillin at 100μg/mL) from 1000X stock solution.
  2. Swipe a single colony from agar plate with a loop/pipette tip.
  3. Place tip in tube, swirling slightly to innoculate the culture.
  4. Incubate overnight in the shaker at 37°C and 250rpm.
  5. The next day the 3mL cultures can be used for DNA isolation or stored in the fridge for up to a week.

Freeze Bacteria

  1. Freezing media is a 50% glycerol solution in dH2O. If none is available in the bacteriological fridge dissolve 10mL of 100% Glycerol (highly viscous) in 10mL of dH2O and store in the fridge.
  2. Use the 1.8mL Cryovials to freeze bacteria.
  3. You want a 1:1 mixture of freezing media and cell suspension (straight from the 5mL tube). Typically add 700μL cell suspension to 700μL of freezing media. If you plan on isolating plasmid DNA only freeze after you have performed the isolation and use whatever cells are left over.
  4. Place in your own labeled box directly in -80°C freezer. Use these the next time you need to grow bacteria.
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