COLONY SELECTION TECHNIQUES – BLUE/WHİTE SCREENING
Rüveyda AKÇİN, Gebze Technical University, Turkey
AIM
Selection of plasmid-containing bacterial cells.
INTRODUCTION
The rate of transformation of chemically
stimulated competent cells is generally low.
This ratio is generally not over 1 / 10,000.
Therefore, bacterial cells containing plasmids
should be selected. Selection of cells
containing plasmid DNA which is transformed
is made according to antibiotic resistance.
Many E. coli strains are susceptible to
antibiotics.
The method is based on the principle of α-
complementation of the β-galactosidase gene.
β-galactosidase is a protein encoded by the
lacZ gene of the lac operon, and it exists as a
homotetramer in its active state. β-
galactosidase catalyzes lactose. To determine
recombinant plasmids and from non-
recombinant plasmids, the '' β-galactosidase ''
gene was added to the multiple cloning site of
the plasmid vector. If the gene of interest is
bound to plasmid multiple cloning site, then
lactose in the medium will not fragment so that
the integrity of the LacZ gene will degrade.
However, if the gene of interest does not bind
to multiple cloning site of plasmid, then lactose
in the medium is degraded and blue color is
formed. (Figure 1)
Figure 1
Another method is colony PCR. It is a highly
efficient PCR. In this technique, specific primers
recognizing the target gene are used. PCR is
performed using the DNA samples obtained
from positive colonies and these primers. If the
agarose gel gives the required band size then it
means to carry the desired DNA fragment.
MATERIALS
 LB
 Ampicillin (10 mg/ml)
 X-gal solution (20 mg/ml): Dissolve X-gal in
an eppendorf tube to a final concentration
of 20 mg/ml in 100 % DMF. Store the stock
solution at -20 C in the dark. Discard the
stock solution if the color changes
significantly. X-gal should be stored
immediately upon receipt at -20 C.
(5-bromo-4-chloro-indolyl-β-D-
galactopyranoside) X-gal is colorless. It is a
chromogenic agent and is used as lactose
analgesic. When X-gal, β-galactosidase is
synthesized, it is degraded by β-
galactosidase and blue color occurs.
 IPTG (100 mM): Dissolve 0.238 g of IPTG in
8 ml of dH2O. Filter sterilize with a 0.2 µm
syringe filter. Store in 1ml aliquots at -20
C.
(Isopropylthiogalactoside) IPTG is a
stimulator for the β-galactosidase gene. If
IPTG is present in the medium, the enzyme
is synthesized.
METHOD
1. Pour sterile warm LB-agar containing
100 µg/ml ampicillin into four petri
dishes.
2. Dry LB plates in laminar flow for about
20 minutes.
3. Mix 20 μl IPTG (100 mM) and 20 μl X-
gal (20 mg/ml) with 60 μl LB in an
eppendorf tube. Then put this solution
onto an agar plate.
4. Spread evenly on the plate with a
sterile glass spreader.
5. Dry the plate in the laminar flow until
all the fluid has disappeared.
6. Attach a numbered grid on the back
side of each petri plate. X-gal+IPTG+Ampicillin+LB (Master):
7. Inoculate the plates with the bacteria
that are to be tested by the method of
toothpicking. For this; pick each colony
in the transformation plate with a
sterile toothpick, and patch it on an LB-
amp plate first, and then on the second
LB-amp plate containing X-gal and
IPTG.
8. Incubate the plates overnight (12 to 16
hr) at 37 °C.
9. Next day morning, incubate the plates
at 4 °C for several hours. This allows
the blue color to develop fully.
10. Examine the plate to determine the
blue and white colonies and note the
number of the White colonies.

RESULT

LB+Ampicillin (Replica):

Both replica and master groups were made for

control purposes. The pUC19s were made to
show how the blue color should be. The under
construction, from clone with sterile toothpick Quandt, Jürgen, and Michael F. Hynes.
were taken samples. Then master was done "Versatile suicide vectors which allow direct
first and replica was made with the same selection for gene replacement in gram-
toothpick. negative bacteria." Gene 127.1 (1993): 15-21.

Timm, Juliano, Eng Mong Lim, and Brigitte

CONCLUSIONS Gicquel. "Escherichia coli-mycobacteria shuttle
First of all, bacterial transfer of the competent vectors for operon and gene fusions to lacZ: the
cell must be checked. Whether the modified pJEM series." Journal of bacteriology 176.21
plasmids are transferred to competent cells (1994): 6749-6753.
should be checked. There are various control
methods for this. However in this experiment,
blue/white screening was done.

Secondly according to experiment result, blue

color was not observed in LB + ampicillin petri
dishes as it should be because X-gal, a lactose
analogue, is absent from the medium. This
group already made with purpose of control.
On the other hand, blue color formation is
observed in petri dishes containing X-gal and
IPTG. This is because, the cells may have not
received the vector or it can also be
contaminant. However, the pUC19 plasmids
have not been changed. The lacZ genes have
not been cut off. Therefore, beta galactosidase
is stimulated by IPTG and X-gal is degraded to
give blue color.

Except this, white does not always mean

clones. Nevertheless, the best possibility must
be considered. Hence, colony PCR can be
performed to confirm the result. Furthermore,
blue/white screening can also be performed
during the transformation stage. But, it does
not make any difference and has a toxic effect.

Consequently, the result of the experiment can

be done well and with these examples, the next
step can be passed.

REFERENCES

Howard, Nathan S., et al. "Color selection with

a hygromycin-resistance-based Escherichia
coli-mycobacterial shuttle vector." Gene 166.1
(1995): 181-182.