Arruda, P

Cremonese, N

Maron, L G

Menossi, M

BioTechniques

English

Repositorio da Producao Cientifica e Intelectual da Unicamp

Making Colony PCR
Easier by Adding Gel-
Loading Buffer to the
Amplification Reaction
BioTechniques 28:424-426 (March 2000)
Because of its simplicity, PCR per-
formed directly from bacterial colonies
on agar plates has been extensively
used as a first step in the identification
of recombinant clones (1–3,6,7). In-
stead of making DNA minipreps and
restriction digestion from several puta-
tive recombinant colonies, colony PCR
allows the identification of the positive
clones, and so reduces these procedures
to one clone per ligation experiment.
When several ligations are being
performed simultaneously or when the
recombinant frequency is expected to
be low and no blue/white screening is
available, it is necessary to perform
several colony PCR amplifications. We
realized that colony PCR could be
made easier if the gel-loading buffer
was included in the amplification mix
instead of being added individually to
each tube just before the electrophore-
sis. We evaluated the effects of three
widely used gel-loading buffers on the
amplification of targets with different
sizes, from different sources and using
several primer combinations.
Plasmids containing inserts with dif-
ferent sizes and coming from different
sources were introduced into Es-
cherichia coli strain DH5α™ (Life
Technologies, Gaithersburg, MD,
USA) by a calcium chloride method
(5). Bacterial lysate was obtained from
ampicillin-resistant colonies on LB
plates by a slight modification of Güs-
sow and Clackson (2): colonies
(0.5–1.5 mm diameter) were picked
and transferred to 50 µL water, boiled
for 5 min and stored at 4°C until used.
Gel-loading buffers (5) were prepared
as 6× concentrated stocks: for buffer I,
0.25% (wt/vol) bromophenol blue,
0.025% (wt/vol) xylene cyanol FF and
40% (wt/vol) sucrose in water; for
buffer II, 0.25% (wt/vol) bromophenol
blue, 0.025% (wt/vol) xylene cyanol FF
and 15% (wt/vol) Ficoll (Type 400) in
water and for buffer III, 0.25% (wt/vol)
bromophenol blue, 0.025% (wt/vol)
xylene cyanol FF and 30% (vol/vol)
glycerol in water. PCR amplifications
were carried out by mixing 2 µL lysate
with 8 µL of a premixture so that the fi-
nal concentration of each reaction was
50 mM KCl, 1.5 mM MgCl2, 10 mM
Tris-HCl, pH 9.0, 200 µM each dNTP,
0.4 µM of each primer, 0.5 U of Taq
DNA polymerase (Amersham Pharma-
cia Biotech, São Paulo, Brazil) and gel-
loading buffer to final concentrations of
0, 0.25 and 0.5×, respectively. (These
low concentrations still cause DNA
samples to sink into the gel wells.) The
inserts were amplified in 0.2 mL tubes
for 30 cycles at 94°C for 30 s, 55°C for
30 s and 72°C for 2 min, followed by a
7 min final extension at 72°C using a
GeneAmp PCR System 9700 thermal
cycler (PE Biosystems, Foster City,
CA, USA). The reactions were separat-
ed by electrophoresis on a 1% TAE
agarose gel.
To evaluate the effects of the three
gel-loading buffers on target amplifica-
tion efficiency, six reactions were per-
formed in parallel, using 2 µL of non-
diluted lysate and lysate dilutions of
1:100, 1:1000, 1:5000, 1:10 000 and
1:20 000 with water. The DNA con-
struct used, pA14, contains a 359 bp in-
sert corresponding to the 3′ untranslat-
ed region from an aluminum-induced
maize gene (our unpublished results)
cloned in pGEM-T Easy (Promega,
Madison, WI, USA). Figure 1 shows
that colony PCR performed without
gel-loading buffers allowed abundant
target amplification even when the bac-
terial lysate was diluted to 1:1000. The
overall amplification in the presence of
0.25× of buffers I, II or III was similar
to the control reaction (no loading
buffer in the PCR mixture). At a
5000-fold dilution, a faint band was
still observed in both the control and in
the samples containing any of the
gel-loading buffers.
With a 0.5× final concentration of
any of the three buffers, the target was
still amplified in those samples starting
from non-diluted bacterial lysate. How-
ever, in the samples amplified with
buffer I, the decrease in the amplifica-
tion efficiency was higher. The effect of
the 0.5× buffer concentration was clear-
ly seen in the samples amplified from
the lysate dilutions because at a 100-fold
dilution, a faint band could be observed
only with buffer III. At higher dilutions,
no amplification could be detected with
any of the three gel-loading buffers.
To verify the performance of the
loading buffers on the amplification of
larger inserts, a PCR was performed us-
ing the lysate obtained from colonies
transformed with p715 (4). This con-
struct contains a 3046 bp insert that
corresponds to the maize HRGP pro-
moter fused to the GUS coding region
Benchmarks
424 BioTechniques Vol. 28, No. 3 (2000)
Figure 1. Colony PCR from bacterial lysate di-
lutions using electrophoresis loading buffers in
the amplification reaction. PCR amplification
was performed without loading buffer (control) or
using 0.25× or 0.5× final concentration of buffers
I, II and III as indicated on the left. The lysate di-
lutions used in the PCR are indicated over each
lane. PCR amplifications were done using for-
ward (5′-GTAAAACGACGGCCAGT-3′) and re-
verse (5′-AACAGCTATGACCATGATTAC-3′)
primers (MWG-Biotech, Ebersberg, Germany).
Reactions were separated on a 1% agarose gel. 
Figure 2. Amplification of a 3046 bp insert by
colony PCR using three different loading
buffers. Insert amplification was performed as
described in Figure 1, using undiluted bacterial
lysate. Reactions were separated on a 1% agarose
gel. C: control (no loading buffer), 1: 0.25× buffer
I; 2: 0.5× buffer I; 3: 0.25× buffer II; 4: 0.5× buffer
II; 5: 0.25× buffer III and 6:0.5× buffer III.
from E. coli and the 3′ terminator re-
gion from Agrobacterium tumefaciens
cloned in pBluescript KS II (Strata-
gene, La Jolla, CA, USA). All loading
buffers allowed the target amplification
at 0.25 and 0.5× final concentration
(Figure 2). However, at 0.5× final con-
centration, there was a decrease in the
amplification that was more pro-
nounced than that observed with the
359 bp target depicted in Figure 1.
After the 0.25× gel-loading buffers
in the PCR mixture proved to be feasi-
ble even when the bacterial lysate was
diluted 1000-fold, we simplified our
protocol: we added the bacterial colony
directly to the 10 µL of the PCR mix-
ture distributed in the 0.2 mL tubes, ac-
cording to Lee and Cooper (3). The cy-
cling parameters were the same as
already described except for the inclu-
sion of one step at 94°C for 3 min be-
fore carrying out the 30 cycles. This
protocol was used to amplify targets
with intermediate sizes and to use other
primers, as seen in Figure 3, using
0.25× buffer I (buffers II and III worked
as well; results are not shown). Using
Taq DNA polymerases from other
brands or a PCR apparatus requiring
mineral oil on the top of the reaction
mixture was also successful (results not
shown).
In conclusion, our results showed
that the three gel-loading buffers can be
added to the PCR mixture used to am-
plify target inserts from bacterial
colonies, decreasing considerably the
tedious handling of several small 0.2
mL tubes that are currently used in the
most recent thermocyclers. We found
that a 0.25× concentration of any of the
three gel-loading buffers does not de-
crease target amplification, although a
negative effect was observed at a 0.5×
concentration. Finally, by decreasing
the reaction volume to 10 µL, the cost
of the assay is considerably lower.
REFERENCES
1.Dallas-Yang, Q., G. Jiang and F.M. Sladek.
1998. Avoiding false positives in colony PCR.
BioTechniques 24:580-582.
2.Güssow, D. and T. Clackson. 1989. Direct
clone characterization from plaques and
colonies by the polymerase chain reaction.
Nucleic Acids Res. 17:4000.
3.Lee, A.B. and T.A. Cooper. 1995. Improved
direct PCR screen for bacterial colonies:
wooden toothpicks inhibit PCR amplification.
BioTechniques 18:225-226.
4.Menossi, M., J.A. Martínez-Izquierdo and
P. Puigdomènech. 1997. Tissue specific ac-
tivity and hormone control of the promoter of
the gene coding for a maize hydroxyproline-
rich glycoprotein. Plant Sci. 125:189-200. 
5.Sambrook, J., E.F. Fritsch and T. Maniatis.
1989. Molecular Cloning: A Laboratory Man-
ual. CSH Laboratory Press, Cold Spring Har-
bor, NY.
6.Sandhu, G.S., J.W. Precup and B.C. Kline.
1989. Rapid one-step characterization of re-
combinant vectors by direct analysis of trans-
formed Escherichia coli colonies. BioTech-
niques 7:689-690.
7.Zon, L.I., D.M. Dorfman and S.H. Orkin.
1989. The polymerase chain reaction colony
miniprep. BioTechniques 7:696-698.
We thank Dr. M.J. da Silva for his help
with the manuscript and D. Johnson for re-
vising the manuscript. The research in our
laboratory was supported by Grants from
PADCT/CNPq (62.0472/98-7) to M.M. and
from the European Community (ERBIC19-
CT-960089) and FAPESP (94/033581-1) to
P.A. M.M., L.G.M. and N.C.J. are recipients
fellowships from FAPESP, Brazil. Address
correspondence to Marcelo Menossi, Cen-
tro de Biologia Molecular e Engenharia
Genética, C.P. 6109, Universidade Estad-
ual de Campinas, CEP 13083-970. Camp-
inas, SP, Brazil. Internet: menossi@uni-
camp.br
Received 11 June 1999; accepted 19
November 1999.
Marcelo Menossi, Nilson
Cremonese, Jr., Lyza G.
Maron and Paulo Arruda
Universidade Estadual de
Campinas
Campinas, Brazil
Heating Greatly Speeds
Coomassie Blue Staining
and Destaining
BioTechniques 28:426-432 (March 2000)
SDS-PAGE is one of the most pow-
erful and commonly used techniques in
molecular biology. The most frequently
used dye for visualizing proteins is
Coomassie Brilliant Blue R-250 be-
cause of its relatively high sensitivity
(3,6). However, its lengthy processing
time is a major drawback of the stan-
dard Coomassie staining technique.
Sambrook et al. (4) state that a staining
time of at least 4 h is needed, with
destaining overnight. This can be short-
ened to a minimum of 1 h staining time,
but at least several hours of destaining
are needed to start to visualize bands.
In addition, the destaining process is
often incomplete even after overnight
destaining, leaving a light blue back-
ground on the gel. This makes photog-
raphy and quantification of the proteins
in the gel difficult and limits the sensi-
tivity of the method.
We rediscovered a paper published
in 1971 where Fairbanks et al. (1) pro-
posed the use of three staining solu-
tions, each containing 10% acetic acid
and progressively lower concentrations
Benchmarks
426 BioTechniques Vol. 28, No. 3 (2000)
Figure 3. Colony PCR from plasmids contain-
ing inserts differing in size, source and using
different primers. Amplifications were done di-
rectly from bacterial colonies coming from plas-
mids containing: lane 1, a 5046 bp insert corre-
sponding to the pET3d entire sequence (New
England Biolabs, Hitchin, England, UK) plus
part of the coding region from the maize Zmal1
gene (GenBank Accession No. AF031083) us-
ing two gene-specific primers. Lane 2, a 3046 bp
insert from p715 amplified with forward and re-
verse primers; lane 3, the same as in lane 2, using
T3 (5′-AATTAACCCTCACTAAAGGG-3′) and
T7 (5′-TACGACTCACTATAGGGCGA-3′) pri-
mers. Lane 4, a 2124 bp insert containing a plant
virus promoter, the coding region from a Pan-
icum miliaceum gene and the 3′ terminator from
Agrobacterium tumefaciens, using forward and
reverse primers. Lane 5, a 662 bp insert corre-
sponding to a part of a plant virus promoter and
the coding region of Zmal1 gene amplified with
gene-specific primers.


Making Colony Pcr Easier By Adding Gel-loading Buffer To The Amplification Reaction.

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Making Colony Pcr Easier By Adding Gel-loading Buffer To The Amplification Reaction.

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