Using the Yeast Two-Hybrid System to Identify Protein–Protein Interactions - Plant Proteomics: Methods and Protocols - page 236

Table 4
Recommended fi nal concentration of amino acids

Amino acid Final Stock solution mL stock for 1 l of media

Adenine 20 mg/l 2 g/l 10

Histidine 20 mg/l 10 g/l 2

Leucine 60 mg/l 10 g/l 6

Lysine 50 mg/l 10 g/l 5

Methionine 20 mg/l 10 g/l 2

Tryptophan 20 mg/l 10 g/l 2

Uracil 20 mg/l 2 g/l 10

3-AT 2 mM 2 M 1

Stock solutions may be autoclaved or fi lter-sterilized using a. 0.2 μ m fi lter and

stored at room temperature or at 4 °C for up to 1 year

5. Yeast Extract Peptone Dextrose Adenine, (YPDA) is a complete
medium for yeast growth supplemented with adenine. It con-
tains yeast extract (10 g/l), peptone (20 g/l), glucose (20 g/l)
and adenine (20 mg/l). Autoclave. If preparing solid YPDA,
add agar (20 g/l) and then autoclave it.
The media requirements for PJ69-4A strain are summarized in
Table 3 . The recommended fi nal concentrations of amino acids are
shown in Table 4 . The amino acids used to supplement the media,
will depend on the Y2H strain used.

2.4 Yeast
Transformation

Solutions and reagents required for yeast transformation are as fol-
lows (use ultrapure water for all solutions and analytical grade
reagents):

1. 2 mg/ml of salmon-sperm DNA or sheared herring testes DNA
(Clontech) to be used as DNA-carrier. Immediately prior to
use, denature the carrier DNA by boiling for 10 min and cool-
ing it on ice.
2. 50 % PEG 4,000 (Polyethylene glycol, avg. MW = 3,350; Sigma-
Aldrich). Filter-sterilized or autoclaved.
3. 1 M Lithium acetate (Sigma-Aldrich), adjust pH to 7.5 using
diluted acetic acid, and fi lter-sterilized.
4. TE buffer 0.1 M Tris-HCl, 10 mM EDTA, adjust pH to 7.5,
and autoclave.

2.5 Plasmid DNA
and Protein Extraction
from Yeast

1. DNA breakage buffer. 2 % Triton X-100, 1 % SDS, 100 mM
sodium chloride, 10 mM Tris-HCl, pH 8 and 1 mM EDTA.
2. Phenol-chloroform-isoamyl alcohol (25:24:1).