The chromogenic substrate X-gal can be mixed with bacterial cultures, mixed with solubilized top agar and spread on selective culture plates. This experiment is from "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.

Operation method

Screening of bacterial colonies with X-gal and IPTG (alpha complementary)

Principle

The chromogenic substrate X-gal can be mixed with bacterial cultures, mixed with solubilized top agar and spread on selective culture plates.

Materials and Instruments

Recombinant plasmid transformed E.coli
IPTG solution X-gal solution
LB or YT agar plates LB or YT top agar Thermostat blocks Wooden toothpicks or inoculation rings

Move

I. Materials

1. Buffers and solvents

IPTG solution (20%, m/V)

X-gal solution (2%, m/V )



2. Culture medium

LB or YT agar plates with appropriate antimicrobials

LB or YT top agar

3. Specialized equipment

Thermostat adjustable to 45°C

Wooden toothpick or inoculation ring

4. Carriers and strains

Recombinant plasmid transformed E.coli.

II. METHODS

1. Dispense the solubilized top layer of agar into 17X100 mm test tubes and place the tubes in a heating block bath at 45°C for spare use.

For 90 mm plates, use 3 ml of top layer agar; for 150 mm plates, use 7 ml of top layer agar.

2. Remove the first tube from the heating block and quickly add 0.1 ml of a bacterial suspension containing not more than 3000 (90 mm plates) or 10000 (150 mm plates) viable bacteria. cover the tube with a tight cap and invert several times to mix the bacteria in the agar.

3. Open the cap of the tube and add the appropriate amount of X-gal and IPTG (if needed) as shown in the table. Tighten the cap and slowly invert the tube several times to mix the contents.



4. Quickly pour the top layer of dissolved agar into the middle of a solidified agar plate containing the appropriate antimicrobials and rotate the plate to disperse the agar.

5. Repeat steps 2-3 for all samples.

6. Allow the agar to solidify at room temperature, wipe off any condensation on the plate cover, and incubate the plate upside down at 37℃ for 12-16 hours.

7. Remove the plates and place them at 4℃ for a few hours for the colonies to develop color.

8. Identify the clones carrying the recombinant plasmid.

The clone carrying the wild-type plasmid contains β-galactosidase activity and the center of the colony is light blue and the periphery is dark blue.

Clones carrying the recombinant plasmid do not have β-galactosidase activity, and the colonies are creamy white or eggshell blue, sometimes with a pseudo-blue spot in the center of the colony.

Observation of agar plates against a bright yellow background enhances the ability of the eye to discriminate between blue and white colonies.

9. Screening and culturing of clones carrying recombinant plasmids.

Blue or white clones in soft agar can form in different orientations, often looking like distant and tilted stars, regardless of their orientation, and these clones can be easily picked out with an inoculating loop or toothpick by piercing the shallow layers of agar and transferred to media containing appropriate antimicrobials.

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