BACTERIAL TRANSFORMATION
Bacterial transformation is used to introduce plasmid
DNA into competent E. coli cells. There are a
number of different types of competent cells that can
be used depending on the application. These
differences are due to the mutations that have been
introduced to make the cells more amenable to
transformation. The genotype description can be
found in the protocol that accompanies the cells.
However, all competent cells have been treated so
that they are primed to take up DNA. Competent
cells are stored at -80°C and should never be allowed
to warm to room temperature - always thaw cells on
ice and keep them cold until ready for transformation.
There are two transformation methods: chemical and
electrical. Each is described below.
CHEMICAL TRANSFORMATION
By far the most common method of bacterial
transformation in our lab is chemical. The most
common competent cells in use in the lab are SURE
(Stratagene), JM109 (Promega), and HB101
(Promega). These cells are obtained already
competent and frozen (-80°C).
Note: modifications to this procedure may be
required when using other cell types. When using kits
that are supplied with competent cells, always refer to
the kit protocol.
Transformation occurs when the competent cells that
take up the plasmid have an altered phenotype, which
is typically observed as antibiotic resistance (e.g.,
ampicillin, kanamycin, or tetracycline). Therefore
cells that take up the plasmid can be selected using
medium containing the appropriate antibiotic.
Resistant cells are identified as single colonies on the
transformant plate. Because plasmids have their own
origin of replication, they are replicated
independently of the host cell’s chromosome. The
plasmid’s origin of replication determines the number
of copies that are produced within each cell. Most
plasmids used in the lab are termed “high-copy
number” and may have up to 100 or more copies/cell.
SURE Protocol.
On agar plates, each well isolated colony originates
from a single cell. Therefore all the cells in the
colony are considered to be identical (clones).
Several individual clones are generally picked for
expansion so that they can be screened for the
plasmid. Transformation is an inefficient process and
it is important to note that not all cells will actually
take up the plasmid. The growth of non-transformed
cells that may be on the plate is inhibited by adding
the appropriate antibiotic to the medium. If multiple
forms of the plasmid are present in the transformation
mix, as occurs when cloning, not all clones will be
identical. This means selection beyond antibiotic
resistance will be required to screen for the clone that
contains the correct of the plasmid. This second level
of screening is typically carried out using either
restriction enzyme digestion or polymerase chain
reaction.
Add an appropriate amount of plasmid to the cells
(e.g., 500 ng of purified plasmid, 2 µl of a ligation
reaction). Incubate the cells on ice for 30 minutes.
Preheat a water bath to 42°C. Preheat culture medium
(SOC) to 42°C.
Label an appropriate number of sterile snap-cap
plastic tubes (17 x 100 mm) and place on ice to chill.
Remove a vial of competent cells and allow them to
thaw on ice. Never transport the cells unless they
are on ice.
Once thawed, aliquot 20 µl of competent cells to each
pre-chilled tube. Do not pipet the cells up/down or
mix violently (i.e., vortex). The cells may be mixed
by gently stirring with the sterile pipet tip.
Heat shock the cells by placing the tubes in the
heated water bath for 30 seconds. Carefully time the
incubation.
Add 80 µl preheated SOC and place the cells back on
ice for 2 minutes.
Incubate the cells for one hour at 37°C and 225 rpm.
This allows the cells to repair their membranes and
begin expressing the antibiotic resistance gene before
antibiotic is added.
Transfer the entire volume into a puddle in the
middle of an LB/amp plate. Using a sterile spreader
spread the cells over the entire surface.
Place the plate right-side-up in the 37°C incubator.
After 10 minutes, turn the plate over and incubate
overnight.
Remove the plates before satellite colonies appear.
The plates can be stored for several days at 4°C.
ELECTROPORATION
Preparation of electrocompetent cells.
Start a fresh overnight culture of cells. Incubate at
37°C with moderate shaking (225 rpm).
Inoculate a flask of LB 1:200 with the overnight
culture. Grow at 37°C with shaking to an O.D.600 of
0.5-0.6.
Chill the cells in an ice-bath 15 minutes and transfer
to a pre-chilled centrifuge bottle. Spin at 4°C for 20
minutes at 5000 xg. Resuspend the cells in 5 ml icecold sterile water (the cells must be kept cold
throughout the procedure). Pellet the cells at 4°C for
20 minutes at 5000 xg. Repeat the wash. After the
second centrifugation, decant the supernatant and
resuspend the cells by swirling in the residual water.
For immediate use, place the suspension in a sterile
pre-chilled 50 ml conical tube and centrifuge at 4°C
for 10 minutes at 5000 xg. Resuspend in 1-2 ml
sterile ice-cold water to yield approximately 2 x 1011
cells/ml. Aliquot 40-300 µl cells into pre-chilled 1.5
ml tubes.
If the cells are to be frozen, add 40 µl sterile ice-cold
10% glycerol to the cells, mix using the pipet tip, and
centrifuge at 4°C for 10 minutes at 5000 xg. Remove
the supernatant and estimate the pellet volume.
Resuspend the cells in an equal volume of sterile icecold 10% glycerol, and aliquot 40-300 µl into sterile
pre-chilled 1.5 ml tubes. Quick-freeze on dry ice and
store at -80°C.
DNA (5 pg 500 ng) to 40-50 µl electrocompetent
cells and mix using the pipet tip.
Transfer the cells and DNA to a pre-chilled
electroporation cuvette (0.2 cm gap) and incubate on
ice for 5 minutes. Dry the cuvette and insert it into
the electroporator’s cuvette holder. Apply the pulse
by pressing the “Pulse” button.
Remove the cuvette and immediately add 1 ml SOC
(without antibiotics). Transfer the cells to a sterile
culture tube. Incubate the cells for 1 hour at 37°C
with moderate shaking.
Plate up to 100 µl cells onto LB/amp plates and
incubate overnight at 37°C.
E. coli
Strain
Field
Strength
(kV/cm)
Transformation Efficiency
(Transformants/µg DNA)
C600
19
2 x 109
K12
17
3.5 x 109
DH5α
17
3 x 109
DH10B
16.6
4 x 109
Electroporation
was
performed
using
the
Electroporator 2510 (Eppendorf), 1 mm cuvette, 40
µl cells, and 10 pg pUC19.
Source: Application note – Eppendorf Electroporator
2510
Electroporation
Set the electroporator to the desired voltage. E. coli
general require a field strength of 16-19 kV/cm to
obtain maximum transformation efficiency. The
optimum field strength should be determined
empirically.
Cells should be kept on ice following preparation or
thawed on ice it previously frozen. Add 1 µl plasmid
June, 2010