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