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Materials: Sterile LB medium (Luria-Bertani Medium) with or without antibiotic: water 500 ml bacto-tryptone 10 g bacto-yeast extracts 5 g sodium chloride 10 g stir to dissolve pH to 7.0 autoclave to sterilize cool to 50-60 degrees C add antibiotic if needed Agar plate with bacterial colonies: scrape bacteria glycerol stock such as E coli DH5 (LifeTechnologies) or scratch a single colonies from an old agar plate with bacteria colonies that has been stored at 4 degrees C. streak onto an agar plate with or without antibiotic incubate the plate inverted overnight at 37 degrees CSupplies: Shaker at 37 degrees CProcedures: Scratch a single colony from the agar plate with bacterial colonies using a sterile small micropipetter tip (use a flamed forceps to hold the tip). Drop the tip in 2.5 ml of LB with or without antibiotics and shake at about 200 rpm for 4-8 hours at 37 degrees C. Use 0.5 ml of this bacteria culture to inoculate 100 ml of LB with antibiotics. Shake at about 200 rpm overnight at 37 degrees C.Results: Culture turns from transparent to cloudy after overnight growth. The overnight culture can be stored at 4 degrees C for several days while preserving the plasmid DNA integrity.
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