Preparing Overnight Bacterial Culture

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 C

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:

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.