Heat-shock transformation of E. coli strains
(ABK version from Tonny Hansen based on Sambrook et al. 1989)
NUF 2006.4.20
Preparation of competent cells
Needed: Growth medium (LB and suitable antibiotics)
Sterile 0.1 M CaCl2 (4°C)
Sterile 75% v/v glycerol (4°C) (if freeze-storage is needed)
Sterile screw-capped centrifuge tubes (for 50 ml culture) and 1.5-mL tubes
Day 1: In the late afternoon, inoculate 5 mL medium (LB+antibiotics) with a single colony.
Incubate overnight at 37°C.
Day 2: Use sterile, ice-cold containers and keep reagents and cells on ice from step 2.
1. Inoculate 50 mL of prewarmed medium with 1 mL overnight culture. Grow at
37°C until OD 0.8-1 (may take ~2 h with optimally growing strain).
Alternatively, skip the use of an overnight culture. Inoculate 50 mL of prewarmed
medium with a single, large colony from a fresh overnight plate and incubate at 37°C for
~3 h with vigorous shaking (~300 rpm). The viable cell concentration should not exceed
108 cells/mL. Monitor OD600 every 20-30 min and compare with a calibration curve.
(Sambrook et al. 1989)
2. Transfer the 50 mL culture to a cold centrifuge tube and let the tube chill on ice
for 5 min. Spin down the culture (5000 rpm, 10 min, 4°C) and carefully discard
all supernatant; (let the tube stand in an inverted position for 1 min to allow all
liquid to drain off).
3. Resuspend the cells in 25 mL ice-cold 0.1 M CaCl2, and keep on ice for 30 min.
4. Harvest the cells (5000 rpm, 4 min, 4°C), carefully discard all supernatant, and
resuspend cells in 1 mL ice-cold 0.1 M CaCl2. (Or resuspend in a volume
corresponding to the OD in step 1.)
5. Cells are ready for heat-shock transformation (or for freeze-storage). Use
100 μL of cell suspension and 1-10 μL of DNA per transformation.
Transformation efficiency remains, or may even increase the first 12-24 hours, when
the cells are kept in 0.1 M CaCl2 on ice for up to 2 days.
6. For storage at -80°C, add sterile glycerol to a final concentration of 12.5% v/v
and store in batches of 100 μL per 1.5-mL Eppendorfer tube. (Add 200 μL of
cold, sterile 75% v/v glycerol per 1 mL of competent cells.)
Heat-shock transformation
1. If cells are frozen, thaw on ice prior to use.
2. Gently mix 100 μL of cells with 1-10 μL of DNA in a 1.5-mL Eppendorfer tube (preferably
by gentle swirling), and keep on ice for 30 min. Make a control with no added DNA.
3. Rapidly transfer the tubes to a 42°C water bath and incubate for exactly 90 seconds. Do
not shake the tubes.
4. Rapidly transfer the tubes to ice, and chill for 1-2 min.
5. Add 1 mL of prewarmed LB or SOC medium and incubate the tubes at 37°C for 45-60
min. Increased transformation efficiency may be obtained by gentle agitation (200 rpm).
6. Plate out 10 μL and 100 μL on selective plates. Harvest the remaining cells, resuspend in
100 μL LB and similarly plate out on selective plates.
Check transformation with 10 ng of pUC19. Maximum efficiencies are around 107 transformed
colonies per μg of supercoiled plasmid DNA.