Preparing Overnight Bacterial Culture
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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
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add antibiotic if needed
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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.
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streak onto an agar plate with or without antibiotic
- incubate the plate inverted overnight 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.
- 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.
