Transformation

Transformation

The following are different transformation methods used as part of our project

Transformation of TSS competent cells

1. Thaw 100µl of TSS/cells on ice
2. Add 1µl of biobrick DNA, pipette gently to mix(100pg – 1 ng of plasmid in a volume of 1-2µl)
3. Incubate on ice for 30 minutes
4. Add 0.9ml SOC at 4°C
5. Incubate for 1 hour at 37°C in shaker
6. Spread 100 µl onto agar plate, containing appropriate antibiotic (see note ii.)
7. Centrifuge remainder of cells at 200rpm / 5 min in a microfuge
8. Remove all except 50 µl of supernatant: resuspend gently with a pipette
9. Plate on a fresh agar plate, containing antibiotic
10. Invert plates and incubate overnight at 37°C


Notes; i) Antibiotic concentrations in LB:
Ampicillin - 100 µg ml-1
Tetracycline - 10 µg ml-1 in ethanol
Chloramphenicol - 30 µg ml-1 in ethanol
Kanamycin - 30 µg ml-1 in dH2O

ii) Antibiotic concentrations in LB-agar:
Ampicillin - 100 µg ml-1
Tetracycline - 5 µg ml-1 in ethanol
Chloramphenicol - 15 µg ml-1 in ethanol
Kanamycin - 30 µg ml-1 in dH2O+

Transformation of Calcium Chloride competent cells

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 sotre at -80°C.