Biology Reference
In-Depth Information
Table 5.6: Producing Competent
E. coli
and Transforming them with Plasmid DNA
Using CaCl
2
.
Production of competent cells
1. Grow a fresh overnight culture of
E. coli
in Luria-Bertani (LB) broth at 37°C.
2. Dilute the cells 40-fold into 1 liter of fresh medium. Incubate at 37°C with good aeration until their
density produces an absorbency rating at 550 nm of 0.4-0.5.
3. Immediately chill the culture by swirling in a ice-water bath.
4. When the cells are chilled, centrifuge the culture at 4°C at 5000 rpm for 10 minutes.
5. Decant the supernatant and place the pellet in ice.
6. Resuspend the pellet in 500 ml of ice-cold 100 mM CaCl
2
. It is easier to resuspend the pellets if they are vortexed
before the CaCl
2
is added. The cells can be suspended by sucking them up and down in a 25-ml pipet.
7. Once the cells are resuspended, incubate in ice for 30 minutes with occasional swirling.
8. Pellet the cells once again at 5000 rpm for 10 minutes at 4°C.
9. Resuspend in 40 ml of ice-cold 100 mM CaCl
2
and 15% glycerol.
10. Distribute aliquots of 0.2 ml of cells into sterile Eppendorf tubes in ice.
11. Keep in ice at 0-4°C for 12-24 hours. This is essential for maximal competency, although the cells are
competent at this stage.
12. Freeze the tubes in ethanol-dry ice or liquid nitrogen and place immediately at
−
70°C. The cells remain
competent for months if stored at
−
70°C.
Transformation of competent cells
1. Thaw a tube of frozen competent cells at 4°C.
2. Add DNA in buffer.
3. Incubate in ice for 30 minutes.
4. Heat shock for 2-5 minutes in a 42°C water bath.
5. Add 0.4 ml of LB broth at room temperature to each tube and incubate for 1 hour at 37°C.
6. Spread on agar plate with appropriate antibiotics. Incubate plates overnight at 37°C.
(Modified from Berger and Kimmel 1987.)
tetracycline plate based on their location. Many derivatives of pBR322 have been
constructed to fulfill particular cloning goals (
Balbas et al. 1986
).
5.10 Transforming
E. coli
with Plasmids
A plasmid carrying exogenous DNA must be inserted into
E. coli
to amplify, or
clone
,
the DNA. The process of inserting a plasmid into
E. coli
is called bacterial
transfor-
mation
. For many years efforts to transform
E. coli
were unsuccessful, and it was
only in 1970 that transformation methods were developed. The ability to transform
E. coli
required understanding its genetics and having the ability to manipulate the
physiological status of the
E. coli
cells to optimize the transformation reaction.
A simple transformation procedure involves suspending
E. coli
cells that are in
the log phase of their growth cycle in an ice-cold solution containing membrane-
disrupting agents such as polyethylene glycol (PEG), dimethyl sulfoxide (DMSO),
or divalent cations such as calcium chloride (
Table 5.6
). Plasmid DNA is added
