Competency

Preparing Competent Cells

Due to limited success using the Calcium Chloride method, we also utilised the TSS method in our experiments.

Preparing competent cells – Calcium Chloride Method

1. Inoculate one E.coli colony in 5ml of LB medium (liquid) over night.
2. Take 1ml of the overnight culture and add it to 100ml of pre-warm LB liquid in a 1L flask.
3. Incubate until the optical density is about 0.5 at 600 nm (~3 hrs).
4. Aseptically transfer 50ml into a Falcon tube (repeat that step) and store on ice for 10min to cool the cultures to 0°C.
5. Centrifuge at 4000rpm for 10min at 4°C.
6. Take off supernatant and leave the tubes in an inverted position for about one minute to drain the reminder.
7. Resuspend the pellet in 10ml of ice-cold 0.1M CaCl2.
8. Centrifuge at 4000rpm for 10min at 4°C.
9. Take off supernatant and leave the tubes in an inverted position for about one minute to drain the reminder.
10. Resuspend in 2ml of ice-cold CaCl2 (0.1M) for each 50ml of original culture.
11. At this point the cells can be split into aliqouts of 200µl and be frozen at -70°C.

Preparing competent cells – TSS Method

1. Inoculate one E.coli colony in 5ml of LB medium (liquid) over night.
2. Take 1ml of the overnight culture and add it to 100ml of pre-warm LB liquid in a 1L flask.
3. Incubate until the optical density is about 0.5 at 600 nm (~3 hrs).
4. Split 100 ml culture into 25 ml aliqouts and incubate on ice for 10min. All the subsequent steps should be carried out at 4°C and cells should be kept on ice as much as possible.
5. Centrifuge for 10min at 3500rpm (4°C).
6. Remove supernatant carefully by pouring and pipetting of the reminder. Place on ice immediately.
7. Resuspend each pellet in 2.5 ml of chilled TSS buffer.
8. Add 100 µl to Eppendorf tubes on ice. Freeze those aliqouts in a dry ice/ethanol bath and store at -80°C.