Beta-galactosidase Screen

Making X-gal Plates

Introduction
These protocols are used to screen bacterial plates for beta-galactosidase activity using X-Gal (aka blue white screening). Each of these protocols has its advantages and disadvantages so be sure to check which one works best for you.

Materials

 * X-gal Stock solution = 100mg/ml in dimethyl formamaide (DMF)
 * Agar
 * Media of your choice
 * Sterile water

Making X-gal plates

 * This method is used if: you won't be keeping the plates more than a week, and the media has a neutral pH.
 * Colonies may take up to 36 hours to develop on these plates, but all positive colonies will be evenly colored.


 * 1) Make the media according to your normal plate recipe and autoclave.
 * 2) Let the media cool in a 60°C water bath.
 * 3) Add X-gal stock solution at a ratio of 2μL per mL media (also add any antibiotic).
 * 4) Swirl gently and pour immediately.
 * 5) Once solid keep out of light.

Spread onto plates

 * This method is used if: you want to turn an existing plate into an X-gal plate, and the media has a neutral pH.
 * Colonies should develop color overnight, but due to uneven distribution of X-gal, all positive colonies may not be evenly colored.
 * 1) Take a pre-poured plate and make sure it is dry (i.e. no condensation on the media) and solid.
 * 2) Dilute the X-gal stock 1 to 1 with sterile water.
 * 3) Pipette 50μL of this dilution onto the plate (If your plate has condensation on it skip the dilution and pipette 25μL of X-gal stock.
 * 4) Evenly distribute the solution onto the plate using a spreader.
 * 5) Let plate dry for 30mins at 37°C.

Agar Overlay

 * This method is used if: your media has a non-neutral pH, you forgot to do one of the other two protocols, or some undergrad in your lab mislabeled the plates.
 * Color should appear on fully developed colonies in 2-8 hours.
 * 1) Microwave 10ml of water with 0.15g agar for each plate to be screened (e.g. for 10 plates it's 100ml water and 1.5g agar).
 * 2) Let the molten agar cool in a 60°C water bath.
 * 3) Add 2μL X-gal stock for each mL of molten agar (e.g. 200μL for 10 plates).
 * 4) Pour 10 ml over each plate to be assayed.
 * 5) Let solidify
 * 6) Incubate plates in a dark place