Team:Macquarie Australia/Cells
Competent Cells
- A sterilized plastic loop was used to gather 10-12 large (2-3mm in diameter) colonies from the plate. Inoculated to 150mL of SOB medium in a 1L flask and grown overnight at 18-22°C, 200-250 rpm.
- A600 readings were between 0.2-0.8 when harvested. Cells were in mid log phase with A600 ~ 0.5.
- The flask was placed on ice for 10 minutes after being removed from incubator.
- Culture was transferred to a 50mL centrifuge tube and spun at 2,500 x g for 10 minutes at 4°C.
- Supernatant was discarded and the tube was placed on ice.
- Cells were resuspended in 1mL of ice-cold TB buffer, ensuring no clumps of cells remained and kept cold.
- Cold TB-buffer was added to bring up volume up to 1/5th of the original culture volume (~30mL) and mixed gently and inverted 3 times.
- Tube was incubated on ice for 10 minutes and centrifuged at 2,500 x g for 7 minutes at 4°C and the supernatant was discarded.
- Cells were resuspended in ~1/20th of the original culture volume of ice cold TB-buffer.
- 930µL of cell suspension was added to pre-chilled 1.5mL Eppendorf tubes.
- 70µL of DMSO was added to the 930µL cell suspension and mixed gently while placed on ice.
- 100µL of the competent cell/DMSO mixture was added into fresh microcentrifuge tubes, labelled and snapped frozen with liquid nitrogen.
- Steps 10-12 was repeated to the remaining for the rest of the suspension from step 9.
