20. 1 M Tris-HCl, pH 9.1: Dissolve 121.14 g of Tris base
(MW ¼ 121.14) in 800 ml of dH 2 O. Adjust the pH to 9.1
with HCl and add dH 2 O until a total volume of 1 l. The pH of
Tris buffers varies with temperature and concentration; therefore, adjust the pH at room temperature. Autoclave to sterilize
and store at room temperature.
21. 80% glycerol: Dilute glycerol with dH 2 O. Autoclave to sterilize
and store at room temperature.
22. Halt protease inhibitor cocktail (100Â) (ThermoFisher Scientific, catalog no. 87786).
23. 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.
24. LB agar plates with carbenicillin: Add 32.0 g of LB agar (Lennox L agar) (ThermoFisher Scientific, catalog no. 22700) per
liter of dH 2 O into an appropriately sized bottle for autoclaving
and swirl to mix. Autoclave to sterilize and cool to ~50–55
C.
Add carbenicillin to a final concentration of 100 μg/ml and
swirl to mix. Flame the neck of the bottle, pour into petri
dishes, and allow to solidify. Store at 4
C for up to 2 months.
Alternatively, the formulation for 1 l of Lennox L agar is as
follows: 10 g of Bacto tryptone, 5 g of Bacto yeast extract, 5 g
of NaCl (MW ¼ 58.44), and 12 g of agar. Swirl to mix and
autoclave to sterilize.
25. 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.
26. PEG-NaCl precipitation solution: Dissolve 200 g of PEG 8000
and 146.1 g of NaCl (MW ¼ 58.44) in dH 2 O to a total volume
of 1 l. Autoclave to sterilize and store at room temperature.
27. TE buffer (10 mM Tris, 1 mM EDTA), pH 8.0: Prepare stock
solutions of 1 M Tris-HCl (pH 8.0) and 0.5 M EDTA
(pH 8.0). To prepare 1 M Tris-HCl (pH 8.0), dissolve
121.14 g of Tris base (MW ¼ 121.14) in 800 ml of dH 2 O. Adjust the pH to 8.0 with HCl (at room temperature) and add
dH 2 O until a total volume of 1 l. Autoclave to sterilize and
store at room temperature. To prepare 0.5 M EDTA (pH 8.0),
add 93.06 g of EDTA disodium salt (MW ¼ 372.24) to 400 ml
of dH 2 O. Adjust the pH to 8.0 with NaOH—the disodium salt
of EDTA will not fully dissolve until the pH of the solution is
adjusted to pH 8.0. Once dissolved, adjust the volume to
500 ml with dH 2 O. Autoclave to sterilize and store at room
temperature. For 1 l of TE buffer, mix 10 ml of 1 M Tris-HCl
(pH 8.0) and 2 ml of 0.5 M EDTA (pH 8.0) with 988 ml of
sterile ddH 2 O. Store at room temperature.
Isolation of Artificial Binding Proteins
109
(MW ¼ 121.14) in 800 ml of dH 2 O. Adjust the pH to 9.1
with HCl and add dH 2 O until a total volume of 1 l. The pH of
Tris buffers varies with temperature and concentration; therefore, adjust the pH at room temperature. Autoclave to sterilize
and store at room temperature.
21. 80% glycerol: Dilute glycerol with dH 2 O. Autoclave to sterilize
and store at room temperature.
22. Halt protease inhibitor cocktail (100Â) (ThermoFisher Scientific, catalog no. 87786).
23. 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.
24. LB agar plates with carbenicillin: Add 32.0 g of LB agar (Lennox L agar) (ThermoFisher Scientific, catalog no. 22700) per
liter of dH 2 O into an appropriately sized bottle for autoclaving
and swirl to mix. Autoclave to sterilize and cool to ~50–55
C.
Add carbenicillin to a final concentration of 100 μg/ml and
swirl to mix. Flame the neck of the bottle, pour into petri
dishes, and allow to solidify. Store at 4
C for up to 2 months.
Alternatively, the formulation for 1 l of Lennox L agar is as
follows: 10 g of Bacto tryptone, 5 g of Bacto yeast extract, 5 g
of NaCl (MW ¼ 58.44), and 12 g of agar. Swirl to mix and
autoclave to sterilize.
25. 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.
26. PEG-NaCl precipitation solution: Dissolve 200 g of PEG 8000
and 146.1 g of NaCl (MW ¼ 58.44) in dH 2 O to a total volume
of 1 l. Autoclave to sterilize and store at room temperature.
27. TE buffer (10 mM Tris, 1 mM EDTA), pH 8.0: Prepare stock
solutions of 1 M Tris-HCl (pH 8.0) and 0.5 M EDTA
(pH 8.0). To prepare 1 M Tris-HCl (pH 8.0), dissolve
121.14 g of Tris base (MW ¼ 121.14) in 800 ml of dH 2 O. Adjust the pH to 8.0 with HCl (at room temperature) and add
dH 2 O until a total volume of 1 l. Autoclave to sterilize and
store at room temperature. To prepare 0.5 M EDTA (pH 8.0),
add 93.06 g of EDTA disodium salt (MW ¼ 372.24) to 400 ml
of dH 2 O. Adjust the pH to 8.0 with NaOH—the disodium salt
of EDTA will not fully dissolve until the pH of the solution is
adjusted to pH 8.0. Once dissolved, adjust the volume to
500 ml with dH 2 O. Autoclave to sterilize and store at room
temperature. For 1 l of TE buffer, mix 10 ml of 1 M Tris-HCl
(pH 8.0) and 2 ml of 0.5 M EDTA (pH 8.0) with 988 ml of
sterile ddH 2 O. Store at room temperature.
Isolation of Artificial Binding Proteins
109