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Elsevier
23
GENE 03769
SUMMARY
We have re-evaluated the conditions for prepmmg competent Escherichia coil cells and established a simple and efficient
method (SEM)for plasmid transfection. Cells (DH5, JMI09 and HB101) prepared by SEM are extremely competent for
transformation (1-3 x 109 efu//~g of pBR322 DNA), and can be stored in liquid nitrogen for at least 40 days without loss
of competence. Unlike electroporation, transformation using these competent cells is affected minimally by salts in DNA
preparation. These competent cells are particularly useful for construction of high-complexity cDNA libraries with a minimum
expenditure of mRNA.
INTRODUCTION
24
sistently high transformation efficiency is difficult in general.
Moreover, frozen competent cells often rapidly lose their
competence during storage, particularly when the final ligation product in the process of eDNA-library construction
is used for transfection. To circumvent these problems, we
systematically optimized each condition and parameter for
the prepacation of E. coli competent cells.
The aim of the present study was to develop SEM for the
preparation of such cells with an extremely high transforming frequency (1-3 x 10 9 cfu//2g of plasmid DNA).
25
1010
P-
u.
lO 9
v-
g~
07
F-
106
0
10
20
Glycerol
(%)
In our standard transformation protocol, frozen competent cells were thawed at room temperature and transferred to an ice bath as soon as thawing was completed.
Competent cells (0.2 ml) have been dispensed into polypropylene of polystyrene tubes (Greiner, F.R.G.). The
pBR322 DNA (10 pg each in 1 #1 of TE) was then added
to the competent cells and the mixture incubated in an ice
bath for 30 min. The volume of DNA solution (up to 20 #1)
had little influence on transformation frequency (data not
shown). The number of transformants increased in proportion to the amount of DNA up to 10 ng. Afterwards,
10 l o
l.,.f-l-lr,.,,.,I
mm~m~ m
I:
S~IO 9
~,
fm- ".,~I,~i
%mm
~ "m\
g[ ~a lO9
lf~i
.m,
~m
"_/ A
-o~
l=Q-
qa
,)
Q.
h.
. ~
100
.m
l--
1o7
s.o
6.0
pH
7.0
lO
"
20
PIPES(mM)
10 7
20 4o eo
MnCI2(mM)
o 12oo3~4ooo
K C I (raM)
lo 20 ~o 4o
CaCl=(mM)
o.o
o.s
1.~
A soo
Fig. 2. Influences ofpH (a) and concentrations of TB components (b-e) on transformation frequency(cfu/#gpBR322 DNA). One of the parameters in
TB (see section a) was varied. The cells DH5 were grown at 18C and harvested at an A6ooof 0A2. Transformation frequencyat each point was obtained
by duplicate measurements.
Fig. 3. Effectof cell concentration at harvest and growth temperature on transformation frequency(cfu/#g of pBR322 DNA). Cells were grown at 37C
(D) or 18C (m), treated with standard TB and transfected following the standard protocol (see section a). Each point denotes an average value of
duplicate measurements.
26
however, the ratio of transformants to the amount of used
D N A decreased rapidly (data not shown).
To optimize heat-shock conditions, transfected cell suspensions were subjected to a heat pulse at 40 , 42 and
44C for 0-90 s. As shown in Fig. 4a, the highest number
of transformants was yielded at 30 s. The temperature for
the heat pulse was not critical, and even after heat shock at
an extremely high temperature (52C) for 30 s, a similar
transformation efficiency was obtained (Fig. 4b).
1010
>,
0e.
~'~
109
e- Q.
'3~
'
'
le
I-
10 7
~ I
10
"
15
10
DMSO (%)
20
30
40
Days
10 10
(%)
'!
c
Z~'A"Z'-,,,)[
0o
L
C
60
4o
a,
ii
o~
"#"'J'-
lOO
J
o
10 9
11,
\.
.m
I-.
20
10 8
20
"
40
"
61~
"
"
80
Time (second)
30
4~)
50
60
Temperature (c)
Fig. 4. Effectsof time (a) and temperature (b) of heat shock on transformation frequency.Cells(DHS) were grown at 18C, harvested at an A60o
of 0.6I and treated with standard transformation buffer. Effect of time
was assessed at three different temperatures; 40C (1), 42C (1"1)and
44C (O). Average values of duplicate measurements were plotted.
5.0
10.0
15.0
20.0
27
form this competent cell within a twofold difference in
frequency. Competent cells with this size limitation are
generally considered sufficient for constructing eDNA
libraries. Since the majority of mRNA ranges from 1-6 kb,
plasmid vectors of up to 4 kb such as the Okayama-Berg
vector can accomodate 6 k b cDNA inse~*.s without
jeopardizing transformation frequency. Hence, use of competent cells prepared by this method is expected to yield a
cDNA library representing the original population of
mRNA
TE a
TE/3 e
TE/9 f
SEM a
Transformation
frequency
Electroporation b
(cfu/#g of pBR322 DNA) c
1.6 109
1.5 109
1.4 x 109
1.1 109
<1 x 10TM
-h
(j) Conclusions
We have described a protocol for preparing competent
E. coli cells which are extremely efficient for plasmid transfection (1-3 x 109 cfu/#g pBR322 DNA) and can be stored
in liquid nitrogen for more than a month. These competent
cells are best suited for the construction of eDNA libraries.
Using these competent cells, we have constructed a eDNA
library with more than 107 independent colonies with as
little as 1 #g of mRNA. Our method is simple, reliable and
highly reproducible. It works for at least three E. coli
strains (DHS, JMI09 and HB101), and possibly for any
other E. coil strains.
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28
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