Blue-White Select Screening Reagent
Catalog Number B3928
Storage Temperature –20 °C
Product Description
Blue/white color selection is a routine technique
employed by molecular biologists. This technique
simplifies the differentiation between colonies/plaques
that contain a cloning vector without an insert and those
that contain a vector harboring an insert of interest.1-3
The technique is based on vectors such as the pUC
and the M13mp series that carry a fragment of the
β-galactosidase gene encoding an α-fragment of
β-galactosidase.1,2 The exploitation of these vectors
requires the use of a bacteria strain carrying the
complementing gene fragment to allow the assembly of
an active complex.4
Equipment and Reagents Required but Not
Provided
• Solid media dishes
• Competent bacteria capable of Blue-White
selection
• Spreader (inoculation stick)
• Turn table
The transformation of the β-galactosidase gene
fragment containing vectors to the appropriate cells, in
the presence of the β-galactosidase chromogenic
substrate X-gal5 and the inducer IPTG,6 results in the
formation of blue colonies/plaques. Disruption of the
β-galactosidase gene by insertion of a DNA fragment
into the vector’s multiple cloning site results in the loss
of functional β-galactosidase activity. Therefore, the
colonies/plaques bearing a vector containing an insert
will remain white and may be easily distinguished from
those bearing an intact cloning vector.
Storage/Stability
Blue-White Select Screening Reagent is shipped on dry
ice and it is recommended to store the product at
–20 °C, protected from light. Under these conditions the
product is stable for 2 years.
The prepared, ready-to-use Blue-White Select
Screening Reagent enables easy selection of
recombinant bacteria/phages in cloning procedure.
Components
•
Blue-White Select Screening Reagent
5 ml
Catalog Number B3928
Sufficient volume for coating ∼125 solid media
dishes (90 mm diameter)
The solution contains 40 mg/ml Isopropyl
β-D-thiogalactopyranoside (IPTG) and 40 mg/ml
5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside
(X-Gal) in dimethylsulfoxide (DMSO).
Precautions and Disclaimer
This product is for R&D use only, not for drug,
household, or other uses. Please consult the Material
Safety Data Sheet for information regarding hazards
and safe handling practices.
Procedure
1. Completely thaw the solution. Make sure it is a
homogeneous solution prior to use.
2. For colony detection:
• Spread 40 µl of the Blue-White Select
Screening Reagent on the desired bacterial
plate. Wait for 5–10 minutes to ensure the
absorption of the solution.
• Plate the transformed bacteria to be tested and
incubate at the desired temperature until
colonies are formed.
3. For plaque detection:
Add 40 µl of the Blue-White Select Screening
Reagent to the top-agar medium.
Notes:
1. If desired, the Blue-White Select Screening
Reagent may be stored as a DMSO/water solution
at –20 °C. The DMSO must be 80–85% (v/v) of the
solution. Take a portion of the stock solution and
add sufficient water to make up 15–20% of the
volume. For the DMSO/water solution, use
46–48 µl per plate. Blue-White Select Screening
Reagent in DMSO/water may be stored at –20 °C
for up to 6 months.
2. Avoid exposing Blue-White Select Screening
Reagent to light. Such exposure causes the
degradation of the X-Gal and the formation of a
yellow impurity. A faint yellow solution is still
functional. However, a dark yellow solution should
be tested on a trial scale before using on a large
scale.
3. In-frame insertion of small DNA fragments into the
β-galactosidase site may not interfere (or not fully
interfere) with β-galactosidase activity, resulting in
the formation of blue or light-blue recombinant
plasmid bearing bacteria colonies.2
References
1. Current Protocols in Molecular Biology. Vol 1,
Ausubel, F.M. et al., eds., John Wiley & Sons (New
York, NY: 1995) pp. 1.4.1-1.4.5, 1.5.5.
2. Molecular Cloning, A Laboratory Manual, 2nd ed.,
Sambrook et al., eds., Cold Spring Harbor
Laboratory Press (Plainview, NY: 1989), pp. 1.81.9, 1.85-1.86, 4.7-4.12.
3. Gronenborn, B., and Messing, J.N., Nature, 272,
375 (1978).
4. Ullmann, A. et al., J. Mol. Biol., 24, 339 (1967).
5. Horwitz, J.P. et al., J. Med. Chem., 7, 574 (1964).
6. Bioard, D.S. et al., Biochim. Biophys. Acta, 1138,
68 (1992).
ADM,NDH,MAM 04/11-1
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