Transformation of E. coli
Preparation of competent cells.
1. Inoculate bacteria to be transformed into 7.5 ml LB liquid, incubate in shaker at 37oC, 250 rpm
for 2.5 - 3.5 hrs. (until culture becomes slightly turbid).
2. Centrifuge culture at setting 5 or 6 of the clinical centrifuge for 5 min., decant supernatant, add 4 - 7 mL ice cold sterile 50 mM CaCl2, vortex to resuspend cells, place on ice for 30 min.
3. Centrifuge cells in cold room at setting 5 or 6 of the clinical centrifuge for 5 min., decant supernatant, add 0.5 mL ice cold sterile 50 mM CaCl2, vortex to resuspend cells, place on
ice.
Transformation with DNA.
4. Add DNA to a labelled sterile 1.5 mL microfuge tube, place on ice.
- use 0.5 - 1 uL of plasmid preparation
- if using ligation, dilute with 5 - 10 vol. ice cold sterile 50 mM CaCl2.
5. Add competent cells to chilled tube containing DNA, mix gently, incubate on ice for 30
min.
6. Heat shock cells in 42oC heat block for 2 min., add 1 mL LB liquid, mix by inversion, lay on side in 37oC incubator, incubate for 1 - 2 hrs.
7. Plate bacteria onto the appropriate selective medium.
- if transforming a plasmid, spread 5 uL and 50 uL on opposite halves of a
plate.
- if transforming a ligation, centrifuge cells briefly ( 15 sec.), decant most of
supernatant, resuspend cells in remaining supernatant by vortexing, spread entire
mixture onto plate.
notes:
- It is very important that bacteria be harvested while still in the log phase of growth, and be kept ice-cold during the entire procedure.
- While fresh cells are always preferable, competent cells may be stored @ 4oC and be used for up to 4 days.
- If screening for the presence of an insert via à-complementation, spread 50 uL X-gal (5- bromo-4-chloro-3-indolyl -D-galactopyranoside) (20 mg/mL in dimethylformamide (DMF)) and 5 uL IPTG (isopropylthio- -D-galactopyranoside) (100 mg/mL in H2O) onto
plate before step 6. IPTG may be omitted when transforming into E. coli strain
TB1.
Back to: