Research Article

Blue-white screening liquid can eliminate false positives in blue-white colony screening

Published: June 10, 2016
Genet. Mol. Res. 15(2): gmr7925 DOI: https://doi.org/10.4238/gmr.15027925
Cite this Article:
(2016). Blue-white screening liquid can eliminate false positives in blue-white colony screening. Genet. Mol. Res. 15(2): gmr7925. https://doi.org/10.4238/gmr.15027925
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Abstract

Although blue-white screening based on α-complementation has been widely used in the screening of genetically modified bacteria, only a single blue-white screening is typically not enough to eliminate false positives. Sometimes, a secondary blue-white screening for the target colonies is required. In this study, two methods were used to investigate the feasibility of secondary blue-white screening for target colonies on lysogeny broth (LB)-ampicillin agar plates. The first method consisted of covering the target colonies grown on LB-ampicillin plate medium with a sterilized filter paper soaked in a solution of 60 μL 20 mg/mL X-gal and 8 μL 20% IPTG. The second method was that blue and white colonies were randomly selected from the blue-white screening plate medium and then re-streaked onto the blue-white screening medium. The colonies were then treated by two methods and incubated at 37°C for 12 h. The results showed that some of the white colonies treated by the two methods showed results similar to the colonies grown on the blue-white screening medium. These results indicate that the target colonies grown on blue-white screening medium can still be used to carry out a secondary blue-white screening. Thus, a blue-white screening liquid was successfully developed. Using the blue-white screening liquid, false positives can be eliminated directly based on the color of the target colonies. This will greatly improve the screening efficiency of positive clones and has important practical implications.

Although blue-white screening based on α-complementation has been widely used in the screening of genetically modified bacteria, only a single blue-white screening is typically not enough to eliminate false positives. Sometimes, a secondary blue-white screening for the target colonies is required. In this study, two methods were used to investigate the feasibility of secondary blue-white screening for target colonies on lysogeny broth (LB)-ampicillin agar plates. The first method consisted of covering the target colonies grown on LB-ampicillin plate medium with a sterilized filter paper soaked in a solution of 60 μL 20 mg/mL X-gal and 8 μL 20% IPTG. The second method was that blue and white colonies were randomly selected from the blue-white screening plate medium and then re-streaked onto the blue-white screening medium. The colonies were then treated by two methods and incubated at 37°C for 12 h. The results showed that some of the white colonies treated by the two methods showed results similar to the colonies grown on the blue-white screening medium. These results indicate that the target colonies grown on blue-white screening medium can still be used to carry out a secondary blue-white screening. Thus, a blue-white screening liquid was successfully developed. Using the blue-white screening liquid, false positives can be eliminated directly based on the color of the target colonies. This will greatly improve the screening efficiency of positive clones and has important practical implications.

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