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

1. Shaker at 37 degrees C

1. 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).
2. 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.
3. Use 0.5 ml of this bacteria culture to inoculate 100 ml of LB with antibiotics.
4. Shake at about 200 rpm overnight at 37 degrees C.

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.