Plate a bacteriophage library at about 30,000-50,000 pfus per 150mm Petri dish by adding an appropriate amount of bacteriophage to 500µl MgSO4-stored E.coli.Dilute bacteria,if needed(ex.VCS237 to A600=0.5).Incubate for 15 minutes at 37°C without shaking. Plate with 7ml LB top agar on LB agar plates.Once the top agar has solidified,turn the plates upside down and incubate overnight at 37°C.

The next day,cool the plates at 4°C for at least one hour(2 hours works well)to prevent the top agar from lifting off the plate during the blotting process.Follow the Benton-Davis blot protocol for blotting and screening.Expose the film long enough to see the background hybridization on each membrane. (This is typically an overnight exposure with a homologous gene from a closely related species).

Excise positive plaque areas with the broad end of a sterile Pasteur pipet and transfer these agar plugs to 500µl TMG.Number these sequentially.Vortex vigorously to loosen the bacteriophage from the agar,then spin in microfuge for 1 minute to pellet the E.coli and the agar.

Follow theBenton-Davis blot protocol for screening these secondary plaques.Pull 3 positive plaques per plate with the small end of a Pasteur pipet and transfer to 100 µl of TMG.Number these secondary plaques using the number of the primary pfu, followed by a period and the numbers 1 through 3(ex. 21., 21.2,and 21.3 for the primary plaque that was numbered 21).Vortex vigorously and microfuge for 1 minute at room temperature to pellet the agar and bacteria.Dilute an aliquot of one of these supernatants 1:2000 in 5µl of TMG and plate with E.coli as above on a small plate.

The next day,pull 3 of these tertiary,plaque-purified plaques per plate,again with the small end of a Pasteur pipet and transfer each into 50 µl TMG.Number these as 21.1a,21.1b and 21.1c.Vortex and spin as above.Plate a lawn of E.coli on a large plate (you may need more than 1 plate depending on the number of bacteriophage you are screening),mark a grid on the bottom of plate and spot 2µl of these 3 supernatants in the appropriate area.Also spot 2µl of the secondary pfus that were not plaque purified(ex. 21.2 and 21.3).Let the 2µl volume soak into the top agar before inverting plate and transferring to 37°C overnight.Benton-Davis blot and screen these the next day.Discard negative supernatants and proceed to the 1.5 ml high titer lysate protocol to begin large scale work-up of positive bacteriophage.To save time,I often begin the 1.5 ml high titer lysate the same night I plate out the 2 µl spot test of the tertiary plaques.

RECIPES
L Broth Agar
1L of L Broth
15g agar
Autoclave

L Broth Top Agar
Make L Broth Agar except substitute 7.5 g agar/L.

TMG
10mM Tris-HCl,pH7.5
10mM MgSO4
0.1% For 100ml:1 ml of 1M Tris-HCl pH 7;1 ml of 1M MgS04;0.1 g gelatin;QS to 100 ml, and autoclave.