### Materials 1. Glass culture tubes with metal caps and labels - Growth medium, from media room or customized - Glass pipette tubes - Parafilm ### Equipment 1. Vortexer - Fireboy or Bunsen burner - Motorized pipette - Micropipettes and sterile tips ### Procedure For a typical liquid culture, use 5 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. Often bacteria with temperature sensitive mutations need to be grown at 30° instead. - 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). - Take your plates from the warm room. Take an aliquot of the antibiotic(s), if needed, from the freezer, and set it on the bench to thaw. - 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. - Remove the tube cap, flame the top of the culture tube, pipette in 5 ml, flame the top of the tube, and cap it. - Pick up one colony by tapping a small (0.1 μl) pipette tip (held on a pipette) on the surface of the plate. Uncap a tube, flame the top, tip the tube so as to transfer cells from the pipette tip to the surface of the media without touching the inside of the tube with the non-sterile portion of the pipetter, flame, cap. You can also use a sterile toothpick for transfer. - Often people use just a sterile metal loop (sterile by flaming) to place the colony in the tube. This is because flaming assures the sterility of the loop, whereas disposables such as pipette tips and toothpicks can be contaminated, and cannot be flamed on the spot. - Pipette the desired amount of antibiotic into each tube along the wall. Do not put the non-sterile part of the pipette inside the tube and use a new tip for each tube. - Vortex each tube for 1-2 seconds to mix well. - Take the tubes to incubate. Turn the rotating rack off using the dial to decrease the speed. Do not use the switch because the stop will be too abrupt. Add your tubes in a balanced layout. If you have an odd number, use extra empty tubes for balance. Turn the rotation back on to 7 (applies to MIT building 68 5th floor warm room). *Do not forget to turn the rack back on.* - Incubation once again is often at 37°C in an incubator or warm room. - 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.