Preparing Overnight Bacterial Culture

Materials:

1. 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
2. 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 C
3. Shaker at 37 degrees C
   • 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:

1. 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.