Downloaded from http://cshprotocols.cshlp.

org/ at Uppsala Univ BMC and Medical Libraries on June 1, 2020 - Published by
Cold Spring Harbor Laboratory Press

Protocol

The Inoue Method for Preparation and Transformation

of Competent Escherichia coli: “Ultracompetent” Cells
Michael R. Green and Joseph Sambrook

This protocol differs from other transformation procedures in that the bacterial culture is grown at
18˚C rather than the conventional 37˚C. Otherwise, the protocol is unremarkable and follows a fairly
standard course. Under standard laboratory conditions, efficiencies of 1 × 108 to 3 × 108 transformed
colonies/µg of plasmid DNA are typical.

MATERIALS

It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental
Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.

RECIPES: Please see the end of this protocol for recipes indicated by <R>. Additional recipes can be found online at
http://cshprotocols.cshlp.org/site/recipes.

Reagents
DMSO
Oxidation products of DMSO, presumably dimethyl sulfone and dimethyl sulfide, are inhibitors of transformation
(Hanahan 1983). To avoid problems, purchase DMSO of the highest quality.

E. coli strain (e.g., DH5α, XL1-Blue) (as a frozen stock)

Inoue transformation buffer (ITB) <R>
Prepare ITB as directed in Step 1.

LB or SOB medium for initial growth of culture

LB (Luria-Bertani) liquid medium <R>
SOB medium <R>
Plasmid DNA
Store a standard preparation of a plasmid DNA in aliquots and use to measure the competence of batches of
chemically treated E. coli. pUC19, which is commonly used for this purpose, is available from New England Biolabs.

SOB agar plates containing 20 mM MgSO4 and the appropriate antibiotic

Standard SOB plates contain 10 mM MgSO4.

SOB medium <R>

SOB medium is used for growth of culture in preparation for transformation. Prepare three 1-L flasks of 250 mL
each and equilibrate the medium to 18˚C–20˚C before inoculation.

SOC medium <R>

Approximately 1 mL of this medium is needed for each transformation reaction.

From the Molecular Cloning collection, edited by Michael R. Green and Joseph Sambrook.
© 2020 Cold Spring Harbor Laboratory Press
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot101196

225
 Downloaded from http://cshprotocols.cshlp.org/ at Uppsala Univ BMC and Medical Libraries on June 1, 2020 - Published by
Cold Spring Harbor Laboratory Press

M.R. Green and J. Sambrook

Equipment
Ice-water bath
Incubator (37˚C)
Liquid nitrogen
Polypropylene tubes (17 × 100 mm; Falcon 2059), chilled in ice
Shaking incubators (18˚C and 37˚C)
Sorvall R-6 and appropriate rotor
Spectrophotometer
Ultramicrocuvettes to read OD at 600 nm
Water baths (37˚C and 42˚C)

METHOD

Perform all steps in this protocol aseptically.

Preparation of Cells
1. Prepare ITB and chill to 0˚C before use.
Organic contaminants present 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
(Millipore) usually gives good results. If problems should arise, treat the deionized H2O with activated
charcoal before use.
i. Prepare 0.5 M PIPES (pH 6.7) by dissolving 15.1 g of PIPES in 80 mL of pure H2O (Milli-Q
or equivalent). Adjust the pH of the solution to 6.7 with 5 M KOH and then add pure H2O to
bring the final volume to 100 mL. Sterilize the solution by filtration through a disposable
prerinsed Nalgene filter (0.45-µm pore size). Divide into aliquots and store frozen at −20˚C.
ii. Dissolve all of the solutes listed below in 800 mL of pure H2O. Then, add 20 mL of 0.5 M
PIPES (pH 6.7). Adjust the volume of the ITB to 1 L with pure H2O.
Reagent Amount per liter Final concentration
MnCl2·4H2O 10.88 g 55 mM
CaCl2·2H2O 2.20 g 15 mM
KCl 18.65 g 250 mM
PIPES (0.5 M, pH 6.7) 20 mL 10 mM
H2O to 1 L

iii. Sterilize the ITB by filtration through a prerinsed 0.45-µm Nalgene filter. Divide into
aliquots and store at −20˚C.
2. 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).
The doubling time of DH5α growing in SOB medium is
30 min at 37˚C.

3. At
6:00 p.m., use this starter culture to inoculate three 2-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˚C–23˚C, with moderate shaking.
As a rule of thumb, grow cultures in a wide-necked flask whose volume is at least 10× that of the volume of
medium. The doubling time of DH5α growing in SOB medium is 150–240 min at 20˚C.
4. The following morning, read the OD600 of all three cultures. Continue to monitor the OD every
45 min.

226 Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot101196

 Downloaded from http://cshprotocols.cshlp.org/ at Uppsala Univ BMC and Medical Libraries on June 1, 2020 - Published by
Cold Spring Harbor Laboratory Press

Inoue Method for Preparing and Transforming Competent E. coli

5. When the OD600 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.
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.
6. Harvest the cells by centrifugation at 2500g for 10 min at 4˚C.
7. 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.
8. Resuspend the cells very gently in 80 mL of ice-cold ITB.
The cells are best suspended by swirling rather than pipetting or vortexing.

9. Harvest the cells by centrifugation at 2500g for 10 min at 4˚C.

10. Pour off the medium and store the open centrifuge tube on a stack of paper towels for 2 min. Use
a vacuum aspirator to remove any drops of remaining medium adhering to the walls of the
centrifuge tube or trapped in its neck.

Freezing of Competent Cells

11. Resuspend the cells gently in 20 mL of ice-cold Inoue transformation buffer.
12. Add 1.5 mL of DMSO. Mix the bacterial suspension by swirling and then store it in ice for 10 min.
13. Working quickly, dispense aliquots of the suspensions into chilled, sterile microcentrifuge tubes.
Immediately snap-freeze the competent cells by immersing the tightly closed tubes in a bath of
liquid nitrogen. Store the tubes at −80˚C until needed.

Freezing in liquid nitrogen enhances transformation efficiency by approximately fivefold. For most cloning
purposes, 50-μL 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.

Transformation
Before using a preparation of competent cells in an important experiment, measure its transformation efficiency using
known amounts of a standard plasmid preparation as described in Protocol: The Hanahan Method for Preparation
and Transformation of Competent Escherichia coli: High-Efficiency Transformation (Green and Sambrook 2018),
and including all of the appropriate positive and negative controls (see Box 1). Using an E. coli strain such as DH5a, the
Inoue procedure typically generates competent cells with transformation efficiencies of 5 × 107 to 3 × 108 colonies/mg
of pUC19 DNA.

14. 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.
15. Use a chilled, sterile pipette tip to transfer the competent cells to chilled, sterile 17 × 100-mm
polypropylene tubes. Store the cells on ice.
Glass tubes should not be used because they lower the efficiency of transformation by
10-fold.

16. Add the transforming DNA (up to 10 ng per 50 µL 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.
Setup 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.

Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot101196 227

 Downloaded from http://cshprotocols.cshlp.org/ at Uppsala Univ BMC and Medical Libraries on June 1, 2020 - Published by
Cold Spring Harbor Laboratory Press

M.R. Green and J. Sambrook

BOX 1. ESSENTIAL CONTROLS FOR BACTERIAL TRANSFORMATION

In every experiment, it is essential to include positive controls to measure the efficiency of transformation,
and negative controls to eliminate the possibility of contamination and to identify the potential causes
of failure.
Negative Controls
An aliquot of competent cells to which no DNA is added should be carried through the transformation
experiment. The entire aliquot should be plated on a single agar plate containing the appropriate antibiotic
used to select transformants. No bacterial colonies should grow on this plate or on a selective plate that
received no bacteria at all. If any are detected, the following possibilities should be considered.
• The competent cells are contaminated with an antibiotic-resistant strain of bacteria during the experi-
ment. Perhaps one of the solutions/reagents used in the transformation protocol is contaminated.
• The selective plates are defective. Perhaps the antibiotic was omitted altogether from the plates or was
added to agar that was too hot.
• The selective plates are contaminated with an antibiotic-resistant strain of bacteria. In this case, colonies
usually appear both on the surface of the medium and in the agar.
Positive Controls
An aliquot of competent cells should be transformed with a known amount of a standard preparation of
circular superhelical plasmid DNA. This control provides a measure of the efficiency of transformation and
allows a standard for comparison with previous transformation experiments.

17. 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 and
50 mL volumes. Other types of tubes may not yield equivalent results. If scaling up, perform
experiments to measure transformation efficiencies at a range of times and volumes.
18. Rapidly transfer the tubes to an ice bath. Allow the cells to cool for 1–2 min.
19. Add 800 µL 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/min) the cells during the recovery
period. If screening by α-complementation, proceed to Protocol: Screening Bacterial Colonies Using X-Gal
and IPTG: α-Complementation (Green and Sambrook 2019).
20. Transfer the appropriate volume (up to 200 µL/90-mm plate) of transformed competent cells
onto agar SOB medium containing 20 mm MgSO4 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
in a microcentrifuge and then gently resuspend the cell pellet in 100 μL of prewarmed SOC medium by
tapping the sides of the tube.
When selecting for resistance to ampicillin, transformed cells should be plated at low density (<104 colonies/
90-mm plate), and the plates should not be incubated for more than 20 h at 37˚C. The enzyme β-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 μg/mL to 100 μg/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.
21. Store the plates at room temperature until the liquid has been absorbed.
22. Invert the plates and incubate them at 37˚C. Transformed colonies should appear in 12–16 h.

228 Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot101196

 Downloaded from http://cshprotocols.cshlp.org/ at Uppsala Univ BMC and Medical Libraries on June 1, 2020 - Published by
Cold Spring Harbor Laboratory Press

Inoue Method for Preparing and Transforming Competent E. coli

DISCUSSION

At its best, this method for preparing competent E. coli from Inoue et al. (1990) can challenge the
efficiencies achieved by Hanahan (Hanahan 1983; Hanahan et al. 1991; and see Protocol: The
Hanahan Method for Preparation and Transformation of Competent Escherichia coli: High-Effi-
ciency Transformation [Green and Sambrook 2018]). However, under standard laboratory condi-
tions, efficiencies of 1 × 108 to 3 × 108 transformed colonies/µg of plasmid DNA are more typical. The
advantages of the procedure are that it is less finicky and more predictable than the original Hanahan
(1983) method.
Why growing the cells at low temperature should affect the efficiency of transformation is any-
body’s guess. Perhaps the composition or the physical characteristics of bacterial membranes synthe-
sized at 18˚C are more favorable for uptake of DNA or perhaps the phases of the growth cycle that
favor efficient transformation are extended.
Incubating bacterial cultures at 18˚C is a challenge. Most laboratories do not have a shaking
incubator that can accurately maintain a temperature of 18˚C in both summer and winter. One
solution is to place an incubator in a 4˚C cold room and use the temperature control to heat the
incubator to 18˚C. Alternatively, there is almost no loss of efficiency if the cultures are grown at 20˚C–
23˚C, which is the ambient temperature in many laboratories. Cultures incubated at such low
temperatures grow slowly with a doubling time of 2.5–4 h. This can lead to frustration, especially
late at night when it seems that the culture will never reach the desired OD600 of 0.6. The answer to this
problem is to set up cultures in the evening and harvest the bacteria early the following morning. The
procedure works well with several strains of E. coli, in particular, DH5α and XL1-Blue. However, the
high transformation efficiency achieved with these strains may not be as easily achieved with others.

RECIPES

Inoue Transformation Buffer (ITB)

Reagent Quantity (for 1 L) Final concentration

MnCl2·4H2O 10.88 g 55 mM
CaCl2·2H2O 2.20 g 15 mM
KCl 18.65 g 250 mM
PIPES (0.5 M, pH 6.7) 20 mL 10 mM
H2O 800 mL
Dissolve the solutes in 800 mL of pure H2O. Then add 20 mL of 0.5 M PIPES (pH 6.7). Adjust the
volume of the buffer to 1 L with pure H2O. Sterilize by filtration through a prerinsed 0.45 µm Nalgene
filter. Divide into aliquots and store at −20˚C.
Prepare 0.5 M PIPES (pH 6.7) (piperazine-1,2-bis[2-ethanesulfonic acid]) by dissolving 15.1 g of
PIPES in 80 mL of pure H2O (Milli-Q, or equivalent). Adjust the pH of the solution to 6.7 with 5 M
KOH, and then add pure H2O to bring the final volume to 100 mL. Sterilize the solution by filtration
through a disposable pre-rinsed Nalgene filter (0.45-µm pore size). Divide into aliquots and store
frozen at −20˚C.
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 (Millipore) usually gives good results. If problems should arise, treat the deionized H2O with
activated charcoal before use.

Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot101196 229

 Downloaded from http://cshprotocols.cshlp.org/ at Uppsala Univ BMC and Medical Libraries on June 1, 2020 - Published by
Cold Spring Harbor Laboratory Press

M.R. Green and J. Sambrook

LB (Luria-Bertani) Liquid Medium

Reagent Amount to add

H2O 950 mL
Tryptone 10 g
NaCl 10 g
Yeast extract 5g
Combine the reagents and shake until the solutes have dissolved. Adjust the pH to 7.0
with 5 N NaOH (
0.2 mL). Adjust the final volume of the solution to 1 L with H2O.
Sterilize by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle.
For solid medium, see the recipe entitled “Media containing agar or agarose.”

Media Containing Agar or Agarose

Prepare liquid media according to the recipe given. Just before autoclaving,
add one of the following:

Bacto agar (for plates) 15 g/L

Bacto agar (for top agar) 7 g/L
Agarose (for plates) 15 g/L
Agarose (for top agarose) 7 g/L
Sterilize by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle. When the
medium is removed from the autoclave, swirl it gently to distribute the melted agar or
agarose evenly throughout the solution. Be careful! The fluid may be superheated and
may boil over when swirled. Before adding thermolabile substances (e.g., antibiotics),
allow the medium to cool to 50˚C–60˚C, and mix the medium by swirling to avoid
producing air bubbles.
Before pouring the plates, set up a color code (e.g., two red stripes for LB-ampicillin
plates; one black stripe for LB plates, etc.), and mark the edges of the plates with the
appropriate colored markers. Pour plates directly from the flask; allow
30–35 mL of
medium per 90-mm plate. To remove bubbles from the medium in the plate, flame the
surface of the medium with a Bunsen burner before the agar or agarose hardens. When
the medium has hardened completely, invert the plates and store them at 4˚C until
needed.
The plates should be removed from storage 1–2 h before they are used. If the plates
are fresh, they will “sweat” when incubated at 37˚C. When this condensation drops on
the agar/agarose surface, it allows bacterial colonies or bacteriophage plaques to spread
and increases the chances of cross-contamination. This problem can be avoided by
wiping off the condensation from the lids of the plates and then incubating the plates
for several hours at 37˚C in an inverted position before they are used. Alternatively,
remove the liquid by shaking the lid with a single, quick motion. To minimize the
possibility of contamination, hold the open plate in an inverted position while removing
the liquid from the lid.

SOB Medium

Per liter: To 950 mL of deionized H2O, add:

Tryptone 20 g
Yeast extract 5g
NaCl 0.5 g

Shake until the solutes have dissolved. Add 10 mL of a 250 mM solution of KCl. (This
solution is made by dissolving 1.86 g of KCl in 100 mL of deionized H2O.) Adjust the pH

230 Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot101196

 Downloaded from http://cshprotocols.cshlp.org/ at Uppsala Univ BMC and Medical Libraries on June 1, 2020 - Published by
Cold Spring Harbor Laboratory Press

Inoue Method for Preparing and Transforming Competent E. coli

of the medium to 7.0 with 5 N NaOH (
0.2 mL). Adjust the volume of the solution to
1 L with deionized H2O. Sterilize by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on
liquid cycle. Just before use, add 5 mL of a sterile solution of 2 M MgCl2. (This solution is
made by dissolving 19 g of MgCl2 in 90 mL of deionized H2O. Adjust the volume of the
solution to 100 mL with deionized H2O and sterilize by autoclaving for 20 min at 15 psi
[1.05 kg/cm2] on liquid cycle.)
For solid medium, please see the recipe entitled “Media containing agar or agarose.”
<R>

SOC Medium

Per liter: To 950 mL of deionized H2O, add:

Tryptone 20 g
Yeast extract 5g
NaCl 0.5 g
SOC medium is identical to SOB medium, except that it contains 20 mM glucose. To
prepare SOB medium, combine the above ingredients and shake until the solutes have
dissolved. Add 10 mL of a 250 mM solution of KCl. (This solution is made by dissolving
1.86 g of KCl in 100 mL of deionized H2O.) Adjust the pH of the medium to 7.0 with 5 N
NaOH (
0.2 mL). Adjust the volume of the solution to 1 L with deionized H2O. Sterilize
by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle. Just before use, add
5 mL of a sterile solution of 2 M MgCl2. (This solution is made by dissolving 19 g of MgCl2
in 90 mL of deionized H2O. Adjust the volume of the solution to 100 mL with deionized
H2O and sterilize by autoclaving for 20 min at 15 psi [1.05 kg/cm2] on liquid cycle.)
After the SOB medium has been autoclaved, allow it to cool to 60˚C or less. Add 20 mL
of a sterile 1 M solution of glucose. (This solution is made by dissolving 18 g of glucose in
90 mL of deionized H2O. After the sugar has dissolved, adjust the volume of the solution
to 100 mL with deionized H2O and sterilize by passing it through a 0.22-µm filter.)

REFERENCES

Green MR, Sambrook J. 2018. The Hanahan method for preparation and
transformation of competent Escherichia coli: High-efficiency transfor-
mation. Cold Spring Harb Protoc doi: 10.1101/pdb.prot101188.
Green MR, Sambrook J. 2019. Screening bacterial colonies using X-Gal and
IPTG: α-Complementation. Cold Spring Harb Protoc doi: 10.1101/pdb
.prot101329.
Hanahan D. 1983. Studies on transformation of Escherichia coli with plas-
mids. J Mol Biol 166: 557–580.
Hanahan D, Jessee J, Bloom FR. 1991. Plasmid transformation of E. coli and
other bacteria. Methods Enzymol 204: 63–113.
Inoue H, Nojima H, Okayama H. 1990. High efficiency transformation of
Escherichia coli with plasmids. Gene 96: 23–28.

Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot101196 231

 Downloaded from http://cshprotocols.cshlp.org/ at Uppsala Univ BMC and Medical Libraries on June 1, 2020 - Published by
Cold Spring Harbor Laboratory Press

The Inoue Method for Preparation and Transformation of Competent

Escherichia coli: ''Ultracompetent'' Cells
Michael R. Green and Joseph Sambrook

Cold Spring Harb Protoc; doi: 10.1101/pdb.prot101196

Email Alerting Receive free email alerts when new articles cite this article - click here.
Service

Subject Browse articles on similar topics from Cold Spring Harbor Protocols.
Categories
Bacteria (50 articles)
DNA Delivery/Gene Transfer (327 articles)
DNA Delivery/Gene Transfer, general (326 articles)
Microbiology (23 articles)
Molecular Biology, general (1226 articles)
Non-Viral Methods (224 articles)
Plasmids (135 articles)
Vectors (128 articles)

To subscribe to Cold Spring Harbor Protocols go to:

http://cshprotocols.cshlp.org/subscriptions

© 2020 Cold Spring Harbor Laboratory Press