A. Preparation of cells
1. Prepare Inoue transformation buffer (chilled to 0 °C before use).
2. Organic contaminants in the H2O used to prepare transformation
buffers can reduce the efficiency of transformation of competent
bacteria. H2O obtained directly from a well-serviced Milli-Q filtration
system usually gives good results. If problems should arise, treat the
deionized H2O with activated charcoal before use.
a. Prepare 0.5 M PIPES (pH 6.7). Adjust the pH of the solution to 6.7 with 5
M KOH, and then add pure H 2 O to bring the final volume to 100 ml.
Sterilize the solution by filtration through a disposable prerinsed
Nalgene filter. Divide into aliquots and store frozen at -20 °C
b. Prepare Inoue transformation buffer by dissolving all of the solutes
listed below in 800 ml of pure H2O and then add 20 ml of 0.5 M PIPES
(pH 6.7). Adjust the volume of the Inoue transformation buffer to 1 liter
with pure H2O.
c. Sterilize Inoue transformation buffer by filtration through a prerinsed
0.45-mm Nalgene filter. Divide into aliquots and store at -20 °C.
3. Pick a single bacterial colony (2-3 mm in diameter) from a plate that has
been incubated for 16-20 h at 37 °C. Transfer the colony into 25 ml of
LB broth or SOB medium in a 250 ml flask. Incubate the culture for 6-8
h at 37 °C with vigorous shaking (250-300 rpm).
4. At about 6 o'clock in the evening, use this starter culture to inoculate
three 1-L flasks, each containing 250 ml of SOB. The first flask receives
10 ml of starter culture, the second receives 4 ml, and the third receives
2 ml. Incubate all three flasks overnight at 18-22 °C with moderate
shaking.
5. The following morning, read the OD 600 of all three cultures. Continue to
monitor the OD every 45 min.
6. When the OD 600 of one of the cultures reaches 0.55, transfer the culture
vessel to an ice-water bath for 10 min. Discard the two other cultures.
7. The ambient temperature of most laboratories rises during the day and
falls during the night. The number of degrees and the timing of the
drop from peak to trough varies depending on the time of year, the
number of people working in the laboratory at night, and so on.
Because of this variability, it is difficult to predict the rate at which
cultures will grow on any given night. Using three different inocula
increases the chances that one of the cultures will be at the correct
density after an overnight incubation.
8. Harvest the cells by centrifugation at 2,500 x g (3,900 rpm in a Sorvall
GSA rotor) for 10 min at
4 °C.
9. Pour off the medium and store the open centrifuge bottle on a stack of
paper towels for 2 min. Use a vacuum aspirator to remove any drops of
remaining medium adhering to walls of the centrifuge bottle or
trapped in its neck.
10. Resuspend the cells gently in 80 ml of ice-cold Inoue transformation
buffer. The cells are best suspended by swirling rather than pipetting or
vortexing.
11. Harvest the cells by centrifugation at 2,500 x g (3,900 rpm in a Sorvall
GSA rotor) for 10 min at
4 °C.
12. Pour off the medium and store the open centrifuge tube on a stack of
paper towels for 2 min.
13. Use a vacuum aspirator to remove any drops of remaining medium
adhering to the walls of the centrifuge tube or trapped in its neck.
B. Freezing of competent cells
1. Resuspend the cells gently in 20 ml of ice-cold Inoue transformation
buffer.
2. Add 1.5 ml of DMSO. Mix the bacterial suspension by swirling and then
store it in ice for 10 min.
3. Working quickly, dispense aliquots of the suspensions into chilled,
sterile microfuge tubes.
4.Immediately snap-freeze the competent cells by immersing the tightly
closed tubes in a bath of liquid nitrogen. Store the tubes at -70 °C until
needed. Freezing in liquid nitrogen enhances transformation efficiency
by ~5-fold. For most cloning purposes, 50 ml aliquots of the competent-
cell suspension will be more than adequate. However, when large
numbers of transformed colonies are required (e.g., when constructing
cDNA libraries), larger aliquots may be necessary.
5. When needed, remove a tube of competent cells from the -70 °C
freezer. Thaw the cells by holding the tube in the palm of the hand. Just
as the cells thaw, transfer the tube to an ice bath. Store the cells on ice
for 10 min.
6. Use a chilled, sterile pipette tip to transfer the competent cells to
chilled, sterile 17 x 100-mm polypropylene tubes. Store the cells on ice.
Glass tubes should not be used since they lower the efficiency of
transformation by ~10-fold.
C. Transformation
1. Include all of the appropriate positive and negative controls.
2. Add the transforming DNA (up to 25 ng per 50 ml of competent cells) in
a volume not exceeding 5% of that of the competent cells. Swirl the
tubes gently several times to mix their contents. Set up at least two
control tubes for each transformation experiment, including a tube of
competent bacteria that receives a known amount of a standard
preparation of superhelical plasmid DNA and a tube of cells that
receives no plasmid DNA at all. Store the tubes on ice for 30 min.
3. Transfer the tubes to a rack placed in a preheated 42 °C circulating
water bath. Store the tubes in the rack for exactly 90 sec. Do not shake
the tubes. Heat shock is a crucial step. It is very important that the cells
be raised to exactly the right temperature at the correct rate. The
incubation times and temperatures given here have been worked out
using Falcon 2059 tubes. Other types of tubes will not necessarily yield
equivalent results.
4. Rapidly transfer the tubes to an ice bath. Allow the cells to cool for 1-2
min.
5. Add 800 ml of SOC medium to each tube. Warm the cultures to 37 °C in
a water bath, and then transfer the tubes to a shaking incubator set at
37 °C. Incubate the cultures for 45 min to allow the bacteria to recover
and to express the antibiotic resistance marker encoded by the
plasmid. To maximize the efficiency of transformation, gently agitate
(<225 cycles/minute) the cells during the recovery period.
6. Transfer the appropriate volume (up to 200 ml per 90 mm plate) of
transformed competent cells onto agar SOB medium containing 20 mM
MgSO 4 and the appropriate antibiotic. When selecting for resistance to
tetracycline, the entire transformation mixture may be spread on a
single plate (or plated in top agar). In this case, collect the bacteria by
centrifuging for 20 sec at room temperature (RT) in a microfuge, and
then gently resuspend the cell pellet in 100 ml of SOC medium by
tapping the sides of the tube. IMPORTANT Sterilize a bent glass rod by
dipping it into ethanol and then in the flame of a Bunsen burner. When
the rod has cooled to RT, spread the transformed cells gently over the
surface of the agar plate. When selecting for resistance to ampicillin,
transformed cells should be plated at low density (<104colonies per 90
mm plate), and the plates should not be incubated for more than 20 h
at 37 °C. The enzyme b-lactamase is secreted into the medium from
ampicillin-resistant transformants and can rapidly inactivate the
antibiotic in regions surrounding the colonies. Thus, plating cells at high
density or incubating them for long periods of time results in the
appearance of ampicillin-sensitive satellite colonies. This problem is
ameliorated, but not completely eliminated, by using carbenicillin
rather than ampicillin in selective media and increasing the
concentration of antibiotic from 60 mg/ml to 100 mg/ml. The number
of ampicillin-resistant colonies does not increase in linear proportion to
the number of cells applied to the plate, perhaps because of growth-
inhibiting substances released from the cells killed by the antibiotic.
7. Store the plates at RT until the liquid has been absorbed.
8. Invert the plates and incubate them at 37 °C. Transformed colonies
should appear in 12-16 h.
