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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

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.

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 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).
5 ml

Equipment and Reagents Required but Not Provided

  • Solid media dishes
  • Competent bacteria capable of Blue-White selection
  • Spreader (inoculation stick)
  • Turn table

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

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.

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

Materials


     

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. 1.8- 1.9, 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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