Lab Resources

M9 Plates

For 1 liter of Plates:

  1. M9 Salts

    Na2HPO4: 6 gm
    KH2PO4: 3 gm
    NH4Cl: 1 gm
    NaCl: 0.5 gm

    Add salts to 450 mls Milli-Q water, dissolve and then bring volume to 495 mls. Pour into a 1-liter erlenmeyer and cover with 2 layers of Al-foil.

  2. Agar

    Add 15 gm Bacto-agar to 500 mls Milli-Q water in 2 liter erlenmeyer flask, add a medium size stir bar, and cover with Al-foil.
  3. Autoclave M9 Salts and Agar solutions using the 20-minute liquid cycle and after autoclaving, place in a water bath set at 55 °C.
  4. 100× M9 Supplement
  5. 100 mM MgSO4·7H2O: 6.16 gm per 250 mls
    20% glucose: 5 gm per 250 mls
    10 mM CaCl2: 2.5 ml 1 M per 250 mls
    1 mg/ml thiamine-HCl 250 mg per 250 mls

    Prepare 250 mls from concentrated stocks or solids and filter sterilize. Store in 'frig.

  6. 1000× Glutamine (C2984H cells require glutamine)   
    20 mg/ml filter sterilized.
  7. 1000× L-Arginine (DH5α NEB cells require both glutamine & arginine) 
    Stock = 126 mg/ml filter sterilized.
  8. 2000× Iron Supplement = 20 mM FeSO4

For 1 liter to make (~40) plates:

After the autoclaved solutions have cooled to 55 °C, add:
Pour the M9 Salts into the Agar flask
10 mls of M9-Supplement per liter
1 ml glutamine

For antibiotic containing plates:

ampicillin: 100 µg/ml final concentration
spectinomycin: 50 µg/ml final concentration
kanamycin: 25 µg/ml final concentration
chloramphenicol: 25 µg/ml final concentration
tetracycline: 10 µg/ml final concentration
Antibiotic stock solutions are typically at 500× or 1000× concentration in water or ethanol (ampicillin and kanamycin must be sterilized by filtering through a 0.22 µm filter and stored in a sterile tube at -20 °C.  Cloramphenicol or tetracycline can be made up in 70% ethanol and do not need to be filtered.  All should be stored at -20 °C.)

When mixing the antibiotic stock solutions into the hot LB+agar, try to avoid bubbles which will ultimately end up in your plates after pouring.  Spread out a "sleeve" of sterile plastic Petri plates on a level surface and pour enough molten M9+agar into each plate to cover the bottom.  (After pouring but while still molten, small bubbles in the plates can be removed by carefully flaming the surface of the plate.)  After pouring, allow the plates to solidify at room temperature and then to sit overnight also at room temperature to allow excess condensation to evaporate before bagging and storing the plates in the 'frig.

Before use, warm plates to room temperature or incubation temperature for transformations.

Recipe Contributed by John Brandis, Ph.D.

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This page last Updated Fri, February 12, 2010 12:02