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Plating

Overview

Done after electroporation or chemical transformation.

Procedure

  1. Shake at 250 rpm for at least 1 hour at 37°C to allow the expression of the antibiotic resistance gene. Meanwhile, prewarm plates (cover side down) at 37C for 1 hour.
    • If the plates contain a lot of moisture, place them cover side up and open the cover ~1/3 way to allow the plates to dry for 30-45 min.
  2. (Optional) To concentrate the cells, spin down for 2 min at 2400 g, then remove supernatant.
  3. Resuspend the cells by a gentle pipetting, then take 5-50 uL.
    • Plate less for higher efficiency plasmids and vice versa.
  4. Plating:
    1. With a spreader (loop):
      1. Immerse the spreader in ethanol and use flame to sterilize it. Allow 10 s to cool, then touch agar at the edge of a plate (not touching the cells).
      2. Gently touch down the spreader and briefly and evenly spread the cells.
        • Do not continue to spread until the sample has absorbed completely as this is lethal to cells.
        • Rotating the plate might help.
      3. Before inverting the plates, allow the surplus liquid to absorb by leaving the plates on the bench for a few minutes.
    2. With beads: Add 10-20 beads, close the lid and rock the plate a few times.
      • Beads spread cells more evenly and do not require ethanol/flame.
      • This is particularly useful if spreading on multiple plates as you can shake them all at the same time by stacking them together.
  5. Incubate at 37C overnight (cover side down; 15-18 h).
    • Extend incubation time by 1-2 hours for larger colonies.
    • If performing blue/white screening, place the plates at 4ºC for a few hours after the colonies have reached the desired size to enhance color development.
  6. Store the remaining transformation mix at +4°C. Additional cells may be plated out the next day, if desired.

Troubleshooting

  • Few or no colonies:
    • Test with a known plasmid, e.g., pUC19, if the cells are alive and the protocol is working.
      • If not, check antibiotic choice and its concentration, media formulation and its stock, and incubation temperature.
      • Make sure you're treating cells very gently. NEver vortex or mix vigorously. Resuspend by a gentle flick of gentle pipetting. Always keep them on ice. If thawed, do not place back to -70C as efficency might drop severalfold.
    • Purify DNA form excess salts, EDTA, proteins that may impede vector and insert ligation.
  • Small satellite colonies apprear:
    • Plates were incubated for too long. Beta-lactamase is secreted by amp-resistant bacteria and eventually clear a zone around themselves from antibiotic where non-recombinants can grow.
    • Antibiotic degraded (old plates or poured into too hot media)

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