The Inoue Method for Preparation and

Transformation of Competent E. coli: "Ultra

Competent" Cells

Joseph Sambrook
Peter Maccallum Cancer Institute and The University of Melbourne, Australia
avid !" #ussell
University of Te$as South%estern Medical Center, allas
&$cerpted 'rom Molecular Clonin() A Laboratory Manual Third Edition

ABSTRACT

At its best, this method for preparin( competent &" coli from Inoue et
al" *+,,-. can challen(e the e/ciencies achieved by 0anahan *+,12."
0o%ever, under standard laboratory conditions, e/ciencies of + $ +-
1

to 2 $ +-
1
transformed colonies3m( of plasmid 4A are more typical"
The advanta(es of the procedure are that it is less 5nicky, more
reproducible, and therefore more predictable than the ori(inal 0anahan
method"

This protocol di6ers from other procedures in that the bacterial culture
is (ro%n at +17C rather than the conventional 287C" 9ther%ise, the
protocol is unremarkable and follo%s a fairly standard course" !hy
(ro%in( the cells at lo% temperature should a6ect the e/ciency of
transformation is anybody:s (uess" Perhaps the composition or the
physical characteristics of bacterial membranes synthesi;ed at +17C
are more favorable for uptake of 4A, or perhaps the phases of the
(ro%th cycle that favor e/cient transformation are e$tended"
Incubatin( bacterial cultures at +17C is a challen(e" Most laboratories
do not have a shakin( incubator that can accurately maintain a
temperature of +17C summer and %inter" 9ne solution is to place an
incubator in a <7C cold room and use the temperature control to heat
the incubator to +17C" Alternatively, there is almost no loss of
e/ciency if the cultures are (ro%n at =->=27C, %hich is the ambient
temperature in many laboratories" Cultures incubated at these
temperatures (ro% slo%ly %ith a doublin( time of ="? to < hours" This
can lead to frustration, especially late at ni(ht %hen it seems that the
culture %ill never reach the desired 9@-- of -"@" The ans%er to this
problem is to set up cultures in the evenin( and harvest the bacteria
early the follo%in( mornin(" The procedure %orks %ell %ith many
strains of &" coli in common use in molecular clonin(, includin( AB+>
Clue, 0+, JM+-2, JM+-13,, 0?a, and 0C+-+"

MATERIAS

Cu6ers, Solutions, and #ea(ents

MS9
9$idation products of MS9, presumably dimethyl sulfone and
dimethyl sul5de, are inhibitors of transformation *0anahan +,1?." To
avoid problems, purchase MS9 of the hi(hest Duality"
Inoue transformation bu6er *please see Step + for details.
?? mM MnCl=E<0=9 +? mM CaCl=E=0=9 =?- mM FCl+- mM PIP&S *-"?M,
p0 @"8. Chilled to -7C before use"
4ucleic Acids And 9li(onucleotides

Plasmid 4A *recombinant plasmid.

Media

BC or S9C medium for initial (ro%th of culture

BC *Buria>Certani. Medium
Per Biter)To ,?- ml of deioni;ed 0=9 , add)Tryptone +- (Geast &$tract ?
(4aCl +- (Shake until the solutes have dissolved" AdHust the p0 to 8"-
%ith ? 4 4a90 *I-"= ml." AdHust the volume of the solution to + liter
%ith deioni;ed 0=9 " Sterili;e by autoclavin( for =- minutes at +? psi
*+"-? k(3cm
=
. on liDuid cycle"
S9C Medium
Per Biter)To ,?- ml of deioni;ed 0=9 , add)Tryptone =- (Geast &$tract ?
(4aCl -"? (Shake until the solutes have dissolved" Add +- ml of a =?-
mM solution of FCl" *This solution is made by dissolvin( +"1@ ( of FCl in
+-- ml of deioni;ed 0=9 ". Adust the p0 of the medium to 8"- %ith ? 4
4a90 *I-"= ml." AdHust the volume of the solution to + liter %ith
deioni;ed 0=9 " Sterili;e by autoclavin( for =- minutes at +? psi *+"-?
k(3cm
=
. on liDuid cycle" Just before use, add ? ml of a sterile solution of
= M M(Cl=" *This solution is made by dissolvin( +, ( of M(Cl= in ,- ml of
deioni;ed 0=9 " AdHust the volume of the solution to +-- ml %ith
deioni;ed 0=9 and sterili;e by autoclavin( for =- minutes at +? psi
*+"-? k(3cm
=
. on liDuid cycle".
S9C a(ar plates containin( =- mM M(S9< and the appropriate
antibiotic
Standard S9C contains +- mM M(S9<"
S9C medium, for (ro%th of culture to be transformed
Prepare three +>liter Jasks of =?- ml each and eDuilibrate the medium
to +1>=-7C before inoculation"
S9C medium
Appro$imately + ml of this medium is needed for each transformation
reaction" S9C medium is identical to S9C medium e$cept it contains =-
mM (lucose" After the S9C medium has been autoclaved, allo% it to
cool to @-7C or less" Add =- ml of a sterile + M solution of (lucose"
*This solution is made by dissolvin( +1 ( of (lucose in ,- ml of
deioni;ed 0=9 " After the su(ar has dissolved, adHust the volume of the
solution to +-- ml %ith deioni;ed 0=9 and sterili;ed by passin( it
throu(h a -"==>mm 5lter".
Centrifu(es and #otors

Sorvall KSA rotor or eDuivalent
Special &Duipment

BiDuid nitro(en Polypropylene tubes *+8 $ +-- mmL 'alcon =-?,.,
chilled in iceShakin( Incubator *+17C.!ater bath preset to <=7C

MET!"#

IMP9#TA4T All steps in this protocol should be carried out aseptically"

Preparation of Cells
1. Prepare Inoue transformation bu6er *chilled to -7C before use."
9r(anic contaminants in the 0=9 used to prepare transformation
bu6ers can reduce the e/ciency of transformation of competent
bacteria" 0=9 obtained directly from a %ell>serviced Milli>M 5ltration
system *Millipore. usually (ives (ood results" If problems should arise,
treat the deioni;ed 0=9 %ith activated charcoal before use"
a" Prepare -"? M PIP&S *p0 @"8. *pipera;ine>+,=>bisN=>ethanesulfonic
acidO. by dissolvin( +?"+ ( of PIP&S in 1- ml of pure 0=9 *Milli>M, or
eDuivalent." AdHust the p0 of the solution to @"8 %ith ? M F90, and then
add pure 0=9 to brin( the 5nal volume to +-- ml" Sterili;e the solution
by 5ltration throu(h a disposable prerinsed 4al(ene 5lter *-"<?>mm
pore si;e." ivide into aliDuots and store fro;en at >=-7C"
b" Prepare Inoue transformation bu6er by dissolvin( all of the solutes
listed belo% in 1-- ml of pure 0
=
9 and then add =- ml of -"? M PIP&S
*p0 @"8." AdHust the volume of the Inoue transformation bu6er to + liter
%ith pure 0
=
9 "
c" Sterili;e Inoue transformation bu6er by 5ltration throu(h a prerinsed
-"<?>mm 4al(ene 5lter" ivide into aliDuots and store at >=-7C"
2. Pick a sin(le bacterial colony *=>2 mm in diameter. from a plate that
has been incubated for +@>=- hours at 287C" Transfer the colony into =?
ml of BC broth or S9C medium in a =?->ml Jask" Incubate the culture for
@>1 hours at 287C %ith vi(orous shakin( *=?->2-- rpm."
3. At about @ o:clock in the evenin(, use this starter culture to inoculate
three +>liter Jasks, each containin( =?- ml of S9C" The 5rst Jask
receives +- ml of starter culture, the second receives < ml, and the third
receives = ml" Incubate all three Jasks overni(ht at +1>==7C %ith
moderate shakin("
4. The follo%in( mornin(, read the 9@-- of all three cultures" Continue
to monitor the 9 every <? minutes"
5. !hen the 9@-- of one of the cultures reaches -"??, transfer the
culture vessel to an ice>%ater bath for +- minutes" iscard the t%o other
cultures"
The ambient temperature of most laboratories rises durin( the day and
falls durin( the ni(ht" The number of de(rees and the timin( of the drop
from peak to trou(h varies dependin( on the time of year, the number of
people %orkin( in the laboratory at ni(ht, and so on" Cecause of this
variability, it is di/cult to predict the rate at %hich cultures %ill (ro% on
any (iven ni(ht" Usin( three di6erent inocula increases the chances that
one of the cultures %ill be at the correct density after an overni(ht
incubation
6. 0arvest the cells by centrifu(ation at =?--( *2,-- rpm in a Sorvall
KSA rotor. for +- minutes at <7C"
7. Pour o6 the medium and store the open centrifu(e bottle on a stack
of paper to%els for = minutes" Use a vacuum aspirator to remove any
drops of remainin( medium adherin( to %alls of the centrifu(e bottle or
trapped in its neck"
8. #esuspend the cells (ently in 1- ml of ice>cold Inoue transformation
bu6er" The cells are best suspended by s%irlin( rather than pipettin( or
vorte$in("
9. 0arvest the cells by centrifu(ation at =?--( *2,-- rpm in a Sorvall
KSA rotor. for +- minutes at <7C"
10. Pour o6 the medium and store the open centrifu(e tube on a stack of
paper to%els for = minutes" Use a vacuum aspirator to remove any drops
of remainin( medium adherin( to the %alls of the centrifu(e tube or
trapped in its neck"
$ree%in& of Competent Cells
11. #esuspend the cells (ently in =- ml of ice>cold Inoue transformation
bu6er"
12. Add +"? ml of MS9" Mi$ the bacterial suspension by s%irlin( and
then store it in ice for +- minutes"
13. !orkin( Duickly, dispense aliDuots of the suspensions into chilled,
sterile microfu(e tubes" Immediately snap>free;e the competent cells by
immersin( the ti(htly closed tubes in a bath of liDuid nitro(en" Store the
tubes at >8-7C until needed" 'ree;in( in liDuid nitro(en enhances
transformation e/ciency by I?>fold" 'or most clonin( purposes, ?->ml
aliDuots of the competent>cell suspension %ill be more than adeDuate"
0o%ever, %hen lar(e numbers of transformed colonies are reDuired *e"(",
%hen constructin( c4A libraries., lar(er aliDuots may be necessary"
14. !hen needed, remove a tube of competent cells from the >8-7C
free;er" Tha% the cells by holdin( the tube in the palm of the hand" Just
as the cells tha%, transfer the tube to an ice bath" Store the cells on ice
for +- minutes"
15. Use a chilled, sterile pipette tip to transfer the competent cells to
chilled, sterile +8 $ +-->mm polypropylene tubes" Store the cells on ice"
Klass tubes should not be used since they lo%er the e/ciency of
transformation by I+->fold
Transformation
Include all of the appropriate positive and ne(ative controls
16. Add the transformin( 4A *up to =? n( per ?- ml of competent cells. in
a volume not e$ceedin( ?P of that of the competent cells" S%irl the
tubes (ently several times to mi$ their contents" Set up at least t%o
control tubes for each transformation e$periment, includin( a tube of
competent bacteria that receives a kno%n amount of a standard
preparation of superhelical plasmid 4A and a tube of cells that receives
no plasmid 4A at all" Store the tubes on ice for 2- minutes"
17. Transfer the tubes to a rack placed in a preheated <=7C
circulatin( %ater bath" Store the tubes in the rack for e$actly ,- seconds"
o not shake the tubes" 0eat shock is a crucial step" It is very important
that the cells be raised to e$actly the ri(ht temperature at the correct
rate" The incubation times and temperatures (iven here have been
%orked out usin( 'alcon =-?, tubes" 9ther types of tubes %ill not
necessarily yield eDuivalent results"
18. #apidly transfer the tubes to an ice bath" Allo% the cells to cool
for +>= minutes"
19. Add 1-- ml of S9C medium to each tube" !arm the cultures to
287C in a %ater bath, and then transfer the tubes to a shakin( incubator
set at 287C" Incubate the cultures for <? minutes to allo% the bacteria to
recover and to e$press the antibiotic resistance marker encoded by the
plasmid" To ma$imi;e the e/ciency of transformation, (ently a(itate
*Q==? cycles3minute. the cells durin( the recovery period"
20. Transfer the appropriate volume *up to =-- ml per ,->mm plate.
of transformed competent cells onto a(ar S9C medium containin( =- mM
M(S9< and the appropriate antibiotic" !hen selectin( for resistance to
tetracycline, the entire transformation mi$ture may be spread on a sin(le
plate *or plated in top a(ar." In this case, collect the bacteria by
centrifu(in( for =- seconds at room temperature in a microfu(e, and
then (ently resuspend the cell pellet in +-- ml of S9C medium by
tappin( the sides of the tube" IMP9#TA4T Sterili;e a bent (lass rod by
dippin( it into ethanol and then in the Jame of a Cunsen burner" !hen
the rod has cooled to room temperature, spread the transformed cells
(ently over the surface of the a(ar plate" !hen selectin( for resistance
to ampicillin, transformed cells should be plated at lo% density *Q+-
<

colonies per ,->mm plate., and the plates should not be incubated for
more than =- hours at 287C" The en;yme b>lactamase is secreted into
the medium from ampicillin>resistant transformants and can rapidly
inactivate the antibiotic in re(ions surroundin( the colonies" Thus, platin(
cells at hi(h density or incubatin( them for lon( periods of time results in
the appearance of ampicillin>sensitive satellite colonies" This problem is
ameliorated, but not completely eliminated, by usin( carbenicillin rather
than ampicillin in selective media and increasin( the concentration of
antibiotic from @- m(3ml to +-- m(3ml" The number of ampicillin>
resistant colonies does not increase in linear proportion to the number of
cells applied to the plate, perhaps because of (ro%th>inhibitin(
substances released from the cells killed by the antibiotic
21. Store the plates at room temperature until the liDuid has been
absorbed"
22. Invert the plates and incubate them at 287C" Transformed
colonies should appear in +=>+@ hours"
RE$ERE'CES

0anahan, " +,12" Studies on transformation of Escherichia coli with
plasmids. J. Mol. Biol" ())) ??8>?1-"
0anahan, " +,1?" TechniDues for transformation of E. coli. In DNA
cloning A !ractical Approach *ed" "M" Klover., vol" + pp" +-,>+2?" I#B
Press, 9$ford, United Fin(dom"
Inoue 0", 4oHima 0", and 9kayama 0" +,,-" 0i(h e/ciency
transformation of Escherichia coli with plasmids" Kene *)) =2>=1"