Method: Assay for Phage Containing the Beta-galactosidase Gene

April 9 1990

C. Helms

Purpose:

This assay is used when working with phage vectors carrying the beta-galactosidase gene (often used for immunological screening). If the cloning event disrupts a normally functional copy of the gene in the vector the resulting plaques would appear clear in the assay. If the phages contain a functional beta-galactosidase gene they will form blue rings around their plaques. Any strain which is not an overproducer of beta- galactosidase will work as indicator host bacteria; a single chromosomal copy of the gene is not a problem.

1. Dimethylformamide
2. X-Gal (5-bromo-4-chloro-3-indolyl-beta-d-galactoside)

3 days total:
1. Day 1: 30 minutes
   
2. Day 2: 1 hour
   
3. Day 3: 5 minutes
   

Day 1

1. Pour X-Gal plates.
2. Inoculate 5 ml LBM with indicator bacteria. Grow overnight at 37 degrees C.

Day 2

Plating the phage:

1. Mix 0.1 ml of indicator bacteria with 3 ml of warm (50-55 degrees C) LBM top agar and pour onto X-Gal plate. Allow the top agar to set for at least 10 minutes.
2. Dilute phage to be tested such that only 1 to 25 phage will be contained in a small drop. (e.g. 10 ul). The reason for the small number of phage is so that individual plaques can be assayed. They give a much clearer result than do confluent spots.
3. Spot 1-10 microliters of phage dilutions onto the hardened agar. Hold for 15 minutes on the bench to let spots partially dry then incubate overnight at 37 degrees C.

Day 3

Interpretation of results:

If phages contain a functional copy of the beta-galactosidase gene blue rings will form around their plaques. With time the whole area of the plaque will become blue. Phages which lack the gene function will remain clear. This test is interpreted most easily when single plaques are assayed. Confluent spots of phage lysis will also form blue rings but this can result either from the presence of the beta- galactosidase gene on the phage or from the lysis of host bacteria which contain their own copy of the gene. This "host interference" does not appear when single plaques are assayed.

Solutions:

X-Gal plates:

 	Melt agar in a 500 ml size bottle  	containing 200 ml 3% agar.   	Pour 200 ml 2 X LBM into the 3%  agar  	and mix well.  	Add 0.8 ml dimethylformamide containing 16 mg X-Gal.  	Mix well. Pour plates immediately; makes 12-16 plates.  

References:

Sambrook J. Fritsch E.F. and T. Maniatis.(1989) Molecular Cloning A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press pp.1.83-1.84 2.43-2.53.

J. Miller. (1972) Experiments in Molecular Genetics p. 48. Cold Spring Harbor Labortory Cold Spring Harbor New York.

CRI Laboratory Manual: RFLPs Project (1989).