Harlequin™ LB Agar

HAL 4

Description

A nutritious molecular biology medium containing the novel chromogen CHE-galactoside to enable rapid, safe and unambiguous detection of plasmid transformed bacteria. The CHE-galactoside replaces the traditional X-gal substrate, simplifying the technique as there is no preparation of stock solutions in dimethyl formamide or dimethyl sulphoxide and surface application of the chromogen to the medium. The intense black colour of the colonies gives a sharper contrast between lac- and lac+ colonies, giving improved colony detection compared to blue X-gal stained colonies.

Formula g/litre

Tryptone 10.0
Yeast Extract 5.0
Sodium chloride 10.0
CHE-galactoside 0.3
IPTG 0.03
Ferric ammonium citrate 0.5
Agar 12.0

Method for reconstitution

Weigh 37.8 grams of powder and disperse in 1 litre of deionised water. Swirl to mix and sterilise by autoclaving at 121°C for 15 minutes. Cool to 47°C and add appropriate filter sterilised antibiotic if required. Pour into sterile Petri dishes, allow the medium to set and dry the surface prior to inoculation.

Appearance:
Straw, clear gel.

pH:
7.0 ± 0.2

Minimum QC organisms - ß-gal reaction
Escherichia coli DH5a (ATCC 53868) Lac Z+ve (black)
Escherichia coli DH5a, Lac Z -ve (remains cream)

Storage of Prepared Medium:
Plates – up to 7 days at 2-8°C in the dark.

Inoculation:
Typically surface spread over plate to detect cream colonies indicating disruption of b-complementation. Alternatively, spread for single colonies if required.

Incubation:
37°C aerobically, for 16-18 hours. The colour of the colonies will substantially increase with prolonged incubation (up to 24 hours).

Interpretation:
Examine for the presence of cream colonies, which indicates a successful insertion of the target DNA.

References
Miller, J.H. (1972). Experiments in Molecular Genetics. Cold Spring Harbour Laboratory. Cold Spring Harbour New ,York. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbour Laboratory. Cold Spring Harbour New, York.