Spin for 2 min at 4000 Â g and aspirate the rest of the liquid.
Resuspend the phage pellet in required volume of PBT
(100 μl/well of bait proteins).
4. Wash the preselection plate four times with 200 μl cold PT
buffer and add 100 μl phage library to each well. Incubate for
1 h at 4
C with shaking.
5. Wash the target-coated plates four times with 200 μl cold PT
buffer and transfer the phage solution from the preselection
plate to the target-coated plate. Allow the phages to bind to the
bait proteins for 2 h at 4
C with shaking.
6. Measure the OD 600 for the E. coli OmniMAX culture supplemented with tetracycline. The OD 600 should be 0.6–0.8 at the
time of phage elution. If necessary, dilute the culture with 2YT
medium and allow to bacteria to grow to desired OD 600 .
Ensure that there is no bacterial growth in the E. coli OmniMAX cultures supplemented with carbenicillin or kanamycin
(see Note 5).
7. Remove the unbound phage solution from the bait proteins
and wash the wells 5 times with 200 μl cold PT buffer.
8. Elute bound phage by adding 100 μl of actively growing
(OD 600 0.6-0.8) E. coli OmniMAX to each well. Cover the
plate with a gas-permeable film and incubate for 30 min at
37
C at 200 rpm.
9. Titrate the number of in-phages during the incubation time:
Make dilution series for 10 μl of in-phages using the log phase
E. coli OmniMAX culture. Add 90 μl culture to each well of a
96-well PCR plate and transfer 10 μl in-phages in first row of
wells, mix, then transfer 10 μl to next well and so on. Change
pipette tips between wells. Spot 5 μl of each dilution on
LB/carb plates. Incubate plates overnight at 37
C.
10. Titer the number of out-phages after incubation: Sample 10 μl
of eluted phages for dilution series. Prepare dilution series as
described above, but using 2YT media. Incubate plates overnight at 37
C.
11. Add 10 μl of M13KO7 helper phage (1 Â 10
11 p.f.u./ml) to
each well. Incubate for 45 min at 37
C at 200 rpm.
12. Transfer the phage infected E. coli OmniMAX culture of each
well to 10 ml 2YT supplemented with 10 μl of carbenicillin and
kanamycin, and 0.3 mM IPTG in 50 ml conical tubes. Incubate
overnight at 37
C with shaking at 200 rpm.
13. Coat the desired number of wells of a 96-well MaxiSorp plate
with bait proteins (10 μl protein in 100 μl PBS) and the
matching number of wells with GST/MBP for preselection in
preparation for next round of selection (see day 1, step 1 + 2).
Incubate the MaxiSorp plates at 4
C overnight under gentle
agitation.
54
Susanne Lu ¨ chow et al.
Resuspend the phage pellet in required volume of PBT
(100 μl/well of bait proteins).
4. Wash the preselection plate four times with 200 μl cold PT
buffer and add 100 μl phage library to each well. Incubate for
1 h at 4
C with shaking.
5. Wash the target-coated plates four times with 200 μl cold PT
buffer and transfer the phage solution from the preselection
plate to the target-coated plate. Allow the phages to bind to the
bait proteins for 2 h at 4
C with shaking.
6. Measure the OD 600 for the E. coli OmniMAX culture supplemented with tetracycline. The OD 600 should be 0.6–0.8 at the
time of phage elution. If necessary, dilute the culture with 2YT
medium and allow to bacteria to grow to desired OD 600 .
Ensure that there is no bacterial growth in the E. coli OmniMAX cultures supplemented with carbenicillin or kanamycin
(see Note 5).
7. Remove the unbound phage solution from the bait proteins
and wash the wells 5 times with 200 μl cold PT buffer.
8. Elute bound phage by adding 100 μl of actively growing
(OD 600 0.6-0.8) E. coli OmniMAX to each well. Cover the
plate with a gas-permeable film and incubate for 30 min at
37
C at 200 rpm.
9. Titrate the number of in-phages during the incubation time:
Make dilution series for 10 μl of in-phages using the log phase
E. coli OmniMAX culture. Add 90 μl culture to each well of a
96-well PCR plate and transfer 10 μl in-phages in first row of
wells, mix, then transfer 10 μl to next well and so on. Change
pipette tips between wells. Spot 5 μl of each dilution on
LB/carb plates. Incubate plates overnight at 37
C.
10. Titer the number of out-phages after incubation: Sample 10 μl
of eluted phages for dilution series. Prepare dilution series as
described above, but using 2YT media. Incubate plates overnight at 37
C.
11. Add 10 μl of M13KO7 helper phage (1 Â 10
11 p.f.u./ml) to
each well. Incubate for 45 min at 37
C at 200 rpm.
12. Transfer the phage infected E. coli OmniMAX culture of each
well to 10 ml 2YT supplemented with 10 μl of carbenicillin and
kanamycin, and 0.3 mM IPTG in 50 ml conical tubes. Incubate
overnight at 37
C with shaking at 200 rpm.
13. Coat the desired number of wells of a 96-well MaxiSorp plate
with bait proteins (10 μl protein in 100 μl PBS) and the
matching number of wells with GST/MBP for preselection in
preparation for next round of selection (see day 1, step 1 + 2).
Incubate the MaxiSorp plates at 4
C overnight under gentle
agitation.
54
Susanne Lu ¨ chow et al.