2.2 Screening
Selected Phage Clones
by Phage ELISA
1. 96-well V-bottom deep well plates (Greiner Bio-One, catalog
no. 786201).
2. 2YT medium: Dissolve 16 g of Bacto tryptone, 10 g of Bacto
yeast extract, and 5 g of NaCl (MW ¼ 58.44) in 900 ml of
dH 2 O. Adjust the pH to 7.0 with NaOH and add dH 2 O until a
total volume of 1 l. Autoclave to sterilize and store at room
temperature.
3. Carbenicillin (500Â stock): Dissolve carbenicillin (50 mg/ml)
in ddH 2 O. Filter-sterilize and store at À20
C. Working concentration is 100 μg/ml.
4. M13KO7 helper phage (New England BioLabs).
5. Kanamycin (500Â stock): Dissolve kanamycin (25 mg/ml) in
ddH 2 O. Filter-sterilize and store at À20
C. Working concentration is 50 μg/ml.
6. Pierce Streptavidin-coated, clear, 96-well plates (ThermoFisher
Scientific, catalog no. 15126).
7. PBS: Prepare a 10Â stock by dissolving 80.0 g of NaCl
(MW ¼ 58.44), 2.0 g of KCl (MW ¼ 74.55), 14.4 g of
Na 2 HPO 4 (MW ¼ 141.96) and 2.4 g of KH 2 PO 4
(MW ¼ 136.09) in 800 ml of dH 2 O. Adjust the pH to 7.4
with HCl and add dH 2 O until a total volume of 1 l. Autoclave
to sterilize and store at room temperature. For a 1Â stock,
dilute the 10Â stock with dH 2 O and adjust the pH to 7.4
with HCl.
8. PBST: Dilute the 10Â PBS to 1Â with dH 2 O and 0.1% (v/v)
Tween 20, and adjust to pH 7.4 with HCl.
9. 10Â Casein Blocking Buffer (Sigma-Aldrich, catalog
no. B6429).
10. 2Â Blocking Buffer: Dilute 10Â Casein Blocking Buffer fivefold in PBS.
11. Biotinylated target protein (recommended 0.5–1.0 μg
per well).
12. Biotinylated deselection target (optional) (recommended
0.5–1.0 μg per well).
13. Anti-Fd-Bacteriophage-HRP (Seramun Diagnostica GmbH,
catalog no. A-020-1-HRP).
14. TMB (SeramunBlau
® fast TMB/substrate solution) (Seramun,
catalog no. S-001-TMB).
15. 0.2 M H 2 SO 4 (optional).
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Anna A. S. Tang et al.
Selected Phage Clones
by Phage ELISA
1. 96-well V-bottom deep well plates (Greiner Bio-One, catalog
no. 786201).
2. 2YT medium: Dissolve 16 g of Bacto tryptone, 10 g of Bacto
yeast extract, and 5 g of NaCl (MW ¼ 58.44) in 900 ml of
dH 2 O. Adjust the pH to 7.0 with NaOH and add dH 2 O until a
total volume of 1 l. Autoclave to sterilize and store at room
temperature.
3. Carbenicillin (500Â stock): Dissolve carbenicillin (50 mg/ml)
in ddH 2 O. Filter-sterilize and store at À20
C. Working concentration is 100 μg/ml.
4. M13KO7 helper phage (New England BioLabs).
5. Kanamycin (500Â stock): Dissolve kanamycin (25 mg/ml) in
ddH 2 O. Filter-sterilize and store at À20
C. Working concentration is 50 μg/ml.
6. Pierce Streptavidin-coated, clear, 96-well plates (ThermoFisher
Scientific, catalog no. 15126).
7. PBS: Prepare a 10Â stock by dissolving 80.0 g of NaCl
(MW ¼ 58.44), 2.0 g of KCl (MW ¼ 74.55), 14.4 g of
Na 2 HPO 4 (MW ¼ 141.96) and 2.4 g of KH 2 PO 4
(MW ¼ 136.09) in 800 ml of dH 2 O. Adjust the pH to 7.4
with HCl and add dH 2 O until a total volume of 1 l. Autoclave
to sterilize and store at room temperature. For a 1Â stock,
dilute the 10Â stock with dH 2 O and adjust the pH to 7.4
with HCl.
8. PBST: Dilute the 10Â PBS to 1Â with dH 2 O and 0.1% (v/v)
Tween 20, and adjust to pH 7.4 with HCl.
9. 10Â Casein Blocking Buffer (Sigma-Aldrich, catalog
no. B6429).
10. 2Â Blocking Buffer: Dilute 10Â Casein Blocking Buffer fivefold in PBS.
11. Biotinylated target protein (recommended 0.5–1.0 μg
per well).
12. Biotinylated deselection target (optional) (recommended
0.5–1.0 μg per well).
13. Anti-Fd-Bacteriophage-HRP (Seramun Diagnostica GmbH,
catalog no. A-020-1-HRP).
14. TMB (SeramunBlau
® fast TMB/substrate solution) (Seramun,
catalog no. S-001-TMB).
15. 0.2 M H 2 SO 4 (optional).
110
Anna A. S. Tang et al.