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HomeCloning & ExpressionBlue-White Select™ Screening Reagent

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
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References

1.
Ausubel F. 1995. Current Protocols in Molecular Biology.. Vol 1 pp. 1.4.1-1.4.5, 1.5.5.. New York: John Wiley & Sons.
2.
Sambrook. 1989. Molecular Cloning, A Laboratory Manual. 2 pp. 1.8- 1.9, 1.85-1.86, 4.7-4.12. Plainview, NY: Cold Spring Harbor Laboratory Press .
3.
GRONENBORN B, MESSING J. 1978. Methylation of single-stranded DNA in vitro introduces new restriction endonuclease cleavage sites. Nature. 272(5651):375-377. http://dx.doi.org/10.1038/272375a0
4.
Ullmann A, Jacob F, Monod J. 1967. Characterization by in vitro complementation of a peptide corresponding to an operator-proximal segment of the ?-galactosidase structural gene of Escherichia coli. Journal of Molecular Biology. 24(2):339-343. http://dx.doi.org/10.1016/0022-2836(67)90341-5
5.
Horwitz JP, Chua J, Curby RJ, Tomson AJ, Da Rooge MA, Fisher BE, Mauricio J, Klundt I. 1964. Substrates for Cytochemical Demonstration of Enzyme Activity. I. Some Substituted 3-Indolyl-?-D-glycopyranosides1a. J. Med. Chem.. 7(4):574-575. http://dx.doi.org/10.1021/jm00334a044
6.
Biard DS, James MR, Cordier A, Sarasin A. 1992. Regulation of the Escherichia coli lac operon expressed in human cells. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression. 1130(1):68-74. http://dx.doi.org/10.1016/0167-4781(92)90463-a