Method: Streaking Lambda Phages

April 9, 1990

C. Helms

Principle:

Phage are streaked onto a medium to obtain an independent isolate prior to preparing a new lysate. This is done to reduce the likelihood of working with lysates which have become contaminated, and/or have accumulated mutations.

Time required:

Less than one hour, but time is spread over three days.

Method A Procedure:

Day 1

1. Inoculate 5 ml LBM medium with the appropriate bacterial host, e. g. LE392. Incubate overnight at 37 degrees C.

Day 2

1. Add 0.1 ml of the overnight culture to 3 ml LBM top agar (kept warm at 50 degrees C), mix briefly and pour onto an LBM plate, swirling the plate to evenly spread the mixture. Allow the agar to set on the bench top, then store at 4 degrees C for an hour to firm the surface.
2. Stab a flamed straight platinum wire or a sterile disposable needle into a phage stock or a single plaque and gently streak the wire over the surface of the hardened agar.
3. Invert the plate and incubate overnight at 37 degrees C.

Day 3

1. Examine plates for isolated plaques; store at 4 degrees C.

Method B Procedure:

Day 1

1. Inoculate 5 ml LBM medium with the bacterial host, e. g. LE392. Incubate overnight at 37 degrees C.

Day 2

1. Spot approximately 10 ul lysate (e. g. a chunk of ice from a frozen stock) onto an LBM plate.
2. Mix 100 ul overnight culture cells and 3 ml warm (50 degrees C) top agar, then pour directly onto the phage spot. Swirl rapidly to distribute the top agar and phage over the surface of the plate.
3. Allow the agar to solidify on the bench top. Invert and incubate overnight at 37 degrees C.

Day 3

1. Examine plates for isolated plaques; store at 4 degrees C.

References: none