Lab Resources

Luria Broth ("LB")

For 0.5 liters of Broth:

A) (from "scratch"): in 450 ml Milli-Q water in a 1 liter beaker with a medium stirring bar, add by sprinkling on top of the water while stirring:

5 gm Bacto-peptone or Bacto-tryptone

2.5 gm Bacto-yeast extract

2.5 gm NaCl

1 stirbar

when the solids are completely dissolved, bring the volume to 0.5 liter with Milli-Q water, transfer to a 1 liter Erlenmeyer flask, cover with 2 layers of Al-foil, set in a glass tray, and autoclave for 20 minutes using the LIQUIDS setting. After autoclaving, allow to cool to 65 °C and then store refrigerated.

For 0.5 liter to make (~20) plates

Prepare broth as indicated above but before autoclaving, add 7.5 gm Bacto-agar per 500 ml. The agar will not dissolve, merely settle as a loose suspension, until heated during autoclaving which causes it to melt and then to form a gel upon cooling.

For antibiotic containing plates:

Prepare LB+agar as described above. After autoclaving, allow LB+agar to cool to about 65 °C (best to place bottles in a water bath at 65 °C after cooling somewhat from the autoclave). When about 65 °C, add appropriate antibiotics or mixtures to:

For high copy number plasmids, typical concentratios are:

ampicillin: 100 µg/ml final concentration.
kanamycin: 25 µg/ml final concentration
chloramphenicol: 34 µg/ml final concentration
tetracycline: 10 µg/ml final concentration
spectinomycin: 50 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 LB+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.

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This document last updated Thu, February 11, 2010 15:48