G418 & Puromycin Kill Curves

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G418 and Puromycin Kill Curves

G418 and puromycin can be used to generate stabally transfected cell lines. However, the concentratio of these antibiotics that are required is highly dependent on the cell type being transfected. This wiki entry contains both the protocol for performing a kill curve, as well as concentrations we have found to work for various cell lines.

Stock Solutions:

  • G418 - 50 mg/ml active compound (typically ~70% activity is normal)
    • Dilute in PBS, filter and store at 4C
  • Puromycin - 5 mg/ml
    • Dilute in water, filter, aliquote in 500 ul or 1 ml stocks and freeze

Kill Curve Protocol:

  1. Search the literature for groups which have used the antibiotic to select the cell line being tested. Use this as the middle concentration and test 10X and 100X less & higher concentrations.
  2. Plate cells into a 6-well or 12-well plate, let recover at least 12 hours & grow to ~80% confluency.
  3. Replace the media, adding the antibiotic at the deisred concentrations
  4. Every 2 days replace the media, keeping the antibiotic concentration the same*
  5. The best concentration is the lowest concentration which gives 100% killing at day 3-4 (puromycin) or 7-10 (G418)
  • For simplicity it is best to prepare ahead of time tubes of media with each dilution of G418/puromycing and antibiotic/anti-fungal added.

Working Concentrations

Cell Type [G418] [Puromycin]

5 ug/ml to 10 ug/ml (1:1,000 or 1:500 dilution)
RAW 264.7
10 ug/ml to 20 ug/ml (1:500 or 1:250 dilution)
10 ug/ml to 20 ug/ml (1:500 or 1:250 dilution)

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