8. Selection. Remove the target from the selection well and wash
6Â with 300 μl of PBST. Transfer the phage from pre-pan well
3 to the selection well. If required, add 1 μg of
non-biotinylated deselection target (see Note 3). Cover with
a plate seal and incubate for 2 h at room temperature on the
vibrating platform shaker.
9. Wash the selection well 27Â with 300 μl of PBST, preferably
using an automated plate washer.
10. Elution 1. Add 100 μl of 0.2 M glycine, pH 2.2 to the selection
well and incubate for 10 min at room temperature. Neutralize
by adding 15 μl of 1 M Tris-HCl, pH 9.1, and transfer the
eluted phage to a microcentrifuge tube.
11. Elution 2. Dilute 14 μl of triethylamine with 986 μl of PBS.
Add 100 μl of the diluted triethylamine to the selection well
and incubate for 6 min at room temperature. Neutralize by
adding 50 μl of 1 M Tris-HCl, pH 7, and transfer the eluted
phage to the microcentrifuge tube containing elution 1.
12. Infection of ER2738 cells. Before eluting the phage, set up an
8 ml culture of ER2738 cells for each selection. Dilute the
overnight cultures of ER2738 cells in 2YT medium with
12 μg/ml tetracycline to an OD600 of ~0.2 and incubate at
37
C, 230 rpm for about 1 h until OD600 reaches ~0.6. Add
the eluted phage and incubate for 1 h at 37
C at up to 90 rpm.
Mix at least once during the incubation.
13. Plating the cells. Plate 1 μl of the phage-infected ER2738 cells
(diluted in 100 μl of 2YT media) onto LB agar plates with
100 μg/ml carbenicillin (the selection antibiotic for the phage
library). Centrifuge the remaining culture at 3000 Â g for
5 min. Pour off the supernatant and resuspend the cell pellet
in the residual supernatant. Plate the cells on LB agar with
100 μg/ml carbenicillin. Incubate the plates overnight at
37
C.
Day 3
14. To estimate the phage titer, count the colonies on the plate
containing 1 μl of phage-infected ER2738 cells. Multiply by
8000 to determine the total number per 8 ml of cells (usually
between 0.1 and 2 Â 10
6
).
15. Scrape the cells from the plate containing the remaining culture. To do this, add 5 ml of 2YT medium with 12 μg/ml
tetracycline and scrape using a disposable plastic spreader.
Transfer the scraped cells to a 50 ml centrifuge tube. Use an
additional 2 ml of medium to scrape off any remaining cells and
mix with the cells in the centrifuge tube. Measure the OD600
of a 1/10 dilution to determine the volume required for an
8 ml culture at OD600 0.2.
112
Anna A. S. Tang et al.
6Â with 300 μl of PBST. Transfer the phage from pre-pan well
3 to the selection well. If required, add 1 μg of
non-biotinylated deselection target (see Note 3). Cover with
a plate seal and incubate for 2 h at room temperature on the
vibrating platform shaker.
9. Wash the selection well 27Â with 300 μl of PBST, preferably
using an automated plate washer.
10. Elution 1. Add 100 μl of 0.2 M glycine, pH 2.2 to the selection
well and incubate for 10 min at room temperature. Neutralize
by adding 15 μl of 1 M Tris-HCl, pH 9.1, and transfer the
eluted phage to a microcentrifuge tube.
11. Elution 2. Dilute 14 μl of triethylamine with 986 μl of PBS.
Add 100 μl of the diluted triethylamine to the selection well
and incubate for 6 min at room temperature. Neutralize by
adding 50 μl of 1 M Tris-HCl, pH 7, and transfer the eluted
phage to the microcentrifuge tube containing elution 1.
12. Infection of ER2738 cells. Before eluting the phage, set up an
8 ml culture of ER2738 cells for each selection. Dilute the
overnight cultures of ER2738 cells in 2YT medium with
12 μg/ml tetracycline to an OD600 of ~0.2 and incubate at
37
C, 230 rpm for about 1 h until OD600 reaches ~0.6. Add
the eluted phage and incubate for 1 h at 37
C at up to 90 rpm.
Mix at least once during the incubation.
13. Plating the cells. Plate 1 μl of the phage-infected ER2738 cells
(diluted in 100 μl of 2YT media) onto LB agar plates with
100 μg/ml carbenicillin (the selection antibiotic for the phage
library). Centrifuge the remaining culture at 3000 Â g for
5 min. Pour off the supernatant and resuspend the cell pellet
in the residual supernatant. Plate the cells on LB agar with
100 μg/ml carbenicillin. Incubate the plates overnight at
37
C.
Day 3
14. To estimate the phage titer, count the colonies on the plate
containing 1 μl of phage-infected ER2738 cells. Multiply by
8000 to determine the total number per 8 ml of cells (usually
between 0.1 and 2 Â 10
6
).
15. Scrape the cells from the plate containing the remaining culture. To do this, add 5 ml of 2YT medium with 12 μg/ml
tetracycline and scrape using a disposable plastic spreader.
Transfer the scraped cells to a 50 ml centrifuge tube. Use an
additional 2 ml of medium to scrape off any remaining cells and
mix with the cells in the centrifuge tube. Measure the OD600
of a 1/10 dilution to determine the volume required for an
8 ml culture at OD600 0.2.
112
Anna A. S. Tang et al.