Shreffler:Bacterial Cell Culture

Inoculation of Bacteria for Overnight Culture

Inoculation is the process of introducing a small amount of bacteria to growth medium in order to grow a large culture (e.g. to obtain more of the recombinant DNA that the cell has been transfected with).

Procedure

Note:Use a flame to keep the LB medium sterile

1. Put 3 mL of LB medium into a 15 mL tube
2. Add 1.5 μL carbinicillin (final concentration 50 μg/mL)
3. With a sterile toothpick held by flamed tweezers, pick a colony off the plate and place the toothpick into the 3 mL of medium
4. Place in shaker overnight (approximately 16 hours) at 37°C at a speed of 240 rpm
5. Carefully remove and add 75 μL of carbinicillin to the two 150 mL flasks of LB medium previously prepared
6. For the next overnight culture, add 300 μL of the first overnight culture to each of the flasks (1 to 500 dilution)
7. Place in shaker overnight (approximately 16 hours) at 37°C at 240 rpm

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