Team:Valencia/Culture ecoli

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Culturing E.coli.

Reagents and Materials

• Glass culture tubes.
• LB medium, from media room or customized.
• Glass pipette tubes.
• Antibiotics.
• 3% agar.

Protocol

For a typical liquid culture, use 25 ml of appropriate medium. The amount in each tube does not have to be exact if you are just trying to culture cells for their precious DNA.

1. Streak an agar plate from glycerol stock. Incubate plates until colonies grow.
• Most incubations with E. coli take place at 37°C.
• The reason you need to streak plates is to be certain that you are starting from a single clonal population of cells. In this case, colonies that are picked are assumed to arise from a single cell dividing to form the colony.
• Agar plates are just the standard. There are many different plating media that can be used (i.e., blood agar).
2. Take your plates from the warm room. Take an aliquot of the antibiotics, if needed, from the freezer, and set it on the bench to thaw.
3. Flame a glass pipette, open the bottle of medium and flame the mouth, measure out the amount you need to fill your tubes, flame the cap and recap the bottle as quickly as possible.
4. Remove the tube cap, flame the top of the culture tube, pipette in 5 ml, flame the top of the tube, and cap it.
5. Pick up one colony by tapping a small (0.1 μl) pipette tip (held on a pipette) on the surface of the plate. Then transfer cells from the pipette spreading it on the plate without touching the inside of the plate with the non-sterile portion of the pipetter. You can also use a sterile toothpick for transfer.
6. Put the plates into a stove at 37ºC.
• Turn down the plates in order to keep the water stuck to the agar.
7. Wait overnight or until your cells have reached the desired concentration.
• The amount of time you wait depends on the reason for growing the cells. To miniprep plasmid DNA, an overnight culture is sufficient. However, when doing measurements of protein levels, take care to take readings at the same cell culture density each time.