16. Dilute the appropriate amount of cells in 2YT medium with
12 μg/ml tetracycline for an 8 ml culture at OD600 0.2.
Incubate for about 1 h at 37
C, 230 rpm until an OD600 of
~0.6 is achieved.
17. Add 3.5 Â 10
10 M13KO7 helper phage (multiplicity of infection ~30) to the culture and incubate at 37
C, 90 rpm for 1 h.
18. Add 16 μl of 25 mg/ml kanamycin and incubate overnight at
25
C, 170 rpm.
Day 4
19. Centrifuge the phage-infected cultures at 3500 Â g for 10 min
and transfer the phage-containing supernatant to fresh tubes.
Remove the required amount of phage-containing supernatant
for panning round 2.
20. Add 2 ml of PEG-NaCl precipitation solution to the remaining
supernatant and incubate for at least 1 h or overnight at 4
C.
21. Centrifuge at 4800 Â g for 30 min to pellet the phage. Pour off
the supernatant (blotting the tube on tissue paper to remove all
of the supernatant) and resuspend the pellet in 320 μl of TE
buffer.
22. Transfer to a microcentrifuge tube and centrifuge at
16,000 Â g for 10 min. Transfer the phage-containing supernatant to a fresh tube. Phage can be stored for several weeks at
4
C. For long-term storage, add an equal volume of 80%
glycerol, mix thoroughly and store at À80
C.
3.1.2 Panning Round 2
Magnetic separation and handling using Dynabeads MyOne Streptavidin T1 magnetic beads can easily be automated on a wide variety
of liquid handling platforms. Panning Round 2 has been automated
on a KingFisher Flex magnetic particle processor (ThermoFisher
Scientific, catalog no. 5400630) and is described by Tang et al.
[35]. However, this method describes how the panning can be
done manually using a magnetic separation rack.
Day 1
1. Block the streptavidin-coated magnetic beads. Resuspend the
Dynabeads MyOne Streptavidin T1 magnetic beads thoroughly by vortexing and transfer 20 μl per selection to an
Eppendorf LoBind microcentrifuge tube. Block in a minimum
of 300 μl 2Â Blocking Buffer (or 100 μl 2Â Blocking Buffer
per 20 μl of streptavidin beads). Incubate overnight at room
temperature on a tube rotator to keep the beads in suspension.
2. Set up an overnight culture of ER2738 E. coli cells. Pick a single
colony into 5 ml of 2YT medium with 12 μg/ml tetracycline
and incubate overnight in an orbital incubator at 37
C,
230 rpm. If performing competitive panning, set this up at
the end of day 2.
Isolation of Artificial Binding Proteins
113
12 μg/ml tetracycline for an 8 ml culture at OD600 0.2.
Incubate for about 1 h at 37
C, 230 rpm until an OD600 of
~0.6 is achieved.
17. Add 3.5 Â 10
10 M13KO7 helper phage (multiplicity of infection ~30) to the culture and incubate at 37
C, 90 rpm for 1 h.
18. Add 16 μl of 25 mg/ml kanamycin and incubate overnight at
25
C, 170 rpm.
Day 4
19. Centrifuge the phage-infected cultures at 3500 Â g for 10 min
and transfer the phage-containing supernatant to fresh tubes.
Remove the required amount of phage-containing supernatant
for panning round 2.
20. Add 2 ml of PEG-NaCl precipitation solution to the remaining
supernatant and incubate for at least 1 h or overnight at 4
C.
21. Centrifuge at 4800 Â g for 30 min to pellet the phage. Pour off
the supernatant (blotting the tube on tissue paper to remove all
of the supernatant) and resuspend the pellet in 320 μl of TE
buffer.
22. Transfer to a microcentrifuge tube and centrifuge at
16,000 Â g for 10 min. Transfer the phage-containing supernatant to a fresh tube. Phage can be stored for several weeks at
4
C. For long-term storage, add an equal volume of 80%
glycerol, mix thoroughly and store at À80
C.
3.1.2 Panning Round 2
Magnetic separation and handling using Dynabeads MyOne Streptavidin T1 magnetic beads can easily be automated on a wide variety
of liquid handling platforms. Panning Round 2 has been automated
on a KingFisher Flex magnetic particle processor (ThermoFisher
Scientific, catalog no. 5400630) and is described by Tang et al.
[35]. However, this method describes how the panning can be
done manually using a magnetic separation rack.
Day 1
1. Block the streptavidin-coated magnetic beads. Resuspend the
Dynabeads MyOne Streptavidin T1 magnetic beads thoroughly by vortexing and transfer 20 μl per selection to an
Eppendorf LoBind microcentrifuge tube. Block in a minimum
of 300 μl 2Â Blocking Buffer (or 100 μl 2Â Blocking Buffer
per 20 μl of streptavidin beads). Incubate overnight at room
temperature on a tube rotator to keep the beads in suspension.
2. Set up an overnight culture of ER2738 E. coli cells. Pick a single
colony into 5 ml of 2YT medium with 12 μg/ml tetracycline
and incubate overnight in an orbital incubator at 37
C,
230 rpm. If performing competitive panning, set this up at
the end of day 2.
Isolation of Artificial Binding Proteins
113