Introduction of recombinant plasmid into cells is achieved by the transformation
of competent cells. Competent cells are prepared by treating the cell with a
divalent cation like calcium chloride. Once the cells are made competent, the
plasmid DNA is mixed with the cells. The competent cells are then subjected
to heat shock, which allows the DNA to enter the cells. The cells are then plated
onto a medium containing antibiotics to allow identification of recombinants.
Competent Cells Preparation
The competent cells were prepared by the following methods:
- Calcium chloride method
- Polyethylene glycol 80(x) method
- Calcium chloride method
- Material calcium chloride (50 mM), E. coli cells, ice, 10% sterile glycerol,
bullets, tips, LB
- Agar plate, LB broth, etc.
strain cells were made competent for transformation by treating them
with calcium chloride, as described by Sambrook et al (1989). Bacteria in glycerol
frozen stock were streaked on a LB agar plate using a sterile platinum loop,
grown at 37°C for 16–20 hours. A single isolated colony from the plate was
transferred into 25 mL of LB broth and grown for 16 hours at 37°C with
shaking until the culture reached an A600 nm of 0.4.
The culture was chilled on ice and the cells were harvested by centrifuging
at 4000 rpm for 10 minutes, at 4°C in a refrigerated centrifuge. The cell pellet
was resuspended in 25 mL ice-cold sterile 50 mM calcium chloride and kept
on ice for 20 minutes. Cells were recovered by centrifugation at 4°C as described
above. Finally, the pellet was gently resuspended in 2.5 mL of ice-cold 50 mM
calcium chloride containing 10% sterile glycerol, aliquoted (200 mL each) in
°C for further use.
Transformation of Competent Cells
- Mix 10 mL of ligation mix (or any other plasmid that is to be transformed)
with 100 mL of competent cells and incubate on ice for 1 hour.
- Apply a heat shock at 42°C for 2 minutes.
- Chill the tube to 0°C on ice immediately.
- Add 800 mL of sac medium and incubate at 37°C for 60 minutes with slow
- Prepare LB-agar plates by adding 2 mL ampicillin (50 mg/mL), 10 mL
IPTG, and 10 mL X.
- Stock per mL of melted LB agar.
- Spread 100 mL of transformed cells onto the plates and incubate at 37°C
Screening of Recombinants
- Selection of recombinants is based on the color of the colony.
- For a pUC vector, since the site used for insertion of foreign DNA is
located within the lacZ gene, insertion of foreign DNA is monitored by the
loss of 13-galactosidase activity upon transformation. Cells with the intact
lacZ gene produce functional 13-galactosidase, which converts the colorless
substrates X-gal to blue chromophor in presence of an inducer IPTG and
therefore produce blue colonies. Transformed cells with recombinant plasmid
do not demonstrate 13-galactosidase activity, and therefore, cannot act on
X-gal resulting in the production of white colonies.
- Select white colonies as clones.
Storage of Clones
- Transfer the white colonies one by one onto a fresh LB agar plate containing
ampicillin, IPTG, and X-Gal.
- Incubate the plate at 37°C overnight. This is the master plate of the clones.
- Inoculate the clones from the master plate to 1 mL of LB containing
100 mL of ampicillin individually.
- Incubate the tubes at 37°C overnight with constant shaking.
- Add 150 mL of 100% glycerol to the wells of the microtiter plate.
- Add 850 mL of the overnight grown culture to glycerol in a microtiter
plate. Use 1 well per clone. Mix well.
- Freeze the plate, cover, and store at –70°C.
- The competent cells prepared may be stored frozen at –70°C without loss
of activity for long time. Store in small aliquots and take out a fresh tube
and use for transformation whenever required.
- The procedure for plating is given for plasmids with lacZ as an insertional
inactivation marker and ampicillin as a selection marker. For plasmids
with other markers, prepare the plating media accordingly.
Count white colonies as recombinant transformants and test for insert. Calculate
the transformation efficiency in terms of the number of colony-forming units
(CFU) per microgram of transforming DNA as follows:
CFU on Plate
CFU/yg = _____________× 101 × dilution factor ng plasmid DNA used