2.6 Yeast
Transformation
1. Wild-type and PBM mutant Y2H bait vectors (pGBT9-GW).
2. YPD liquid medium.
3. Sterile ddH 2 O.
4. SC –Trp plates (see Table 2).
5. 10Â TE buffer: 100 mM Tris–HCl, pH 8; and 10 mM EDTA,
pH 8 (autoclave).
6. 10Â lithium acetate (LiAc): 1 M LiAc. Adjust pH to 7.5 with
dilute acetic acid, filter-sterilize on 0.22 μm membrane, and
store at 4
C.
7. 10 mg/mL single-stranded carrier DNA from salmon sperm.
8. PEG–TE–LiAC solution: 50% (w/v) polyethylene glycol 4000
(PEG 4000) in 1Â TE–LiAc solution (autoclave).
9. Dimethyl sulfoxide (DMSO).
2.7 Screening
of the Human PDZome
Library
1. SC -Leu plates.
2. Y2H PDZome array (frozen stock).
3. Wild-type and PBM mutant bait-containing AH109 strains.
4. SC -Leu liquid medium.
5. SC -Trp liquid medium.
6. YPD liquid medium.
7. SC -Leu, -Trp plates.
8. SC -Leu, -Trp, -His plates.
9. 96w polypropylene deep-well plates
10. 96-well U-bottom microplates.
Table 2
Amino acid dropout supplements and selection purposes
Selection purpose
Selective SC medium
Required supplement (8 mL/L)
Prey selection
SC -Leu
Trp, His, Ura
Bait selection
SC -Trp
Leu, His, Ura
Bait autoactivation readout
SC -Trp -His
Leu, Ura
Mating efficiency readout
-Leu -Trp
His, Ura
Y2H interaction readout
-Leu -Trp -His
Ura
Y2H Screening of PDZ Interactions
7
Transformation
1. Wild-type and PBM mutant Y2H bait vectors (pGBT9-GW).
2. YPD liquid medium.
3. Sterile ddH 2 O.
4. SC –Trp plates (see Table 2).
5. 10Â TE buffer: 100 mM Tris–HCl, pH 8; and 10 mM EDTA,
pH 8 (autoclave).
6. 10Â lithium acetate (LiAc): 1 M LiAc. Adjust pH to 7.5 with
dilute acetic acid, filter-sterilize on 0.22 μm membrane, and
store at 4
C.
7. 10 mg/mL single-stranded carrier DNA from salmon sperm.
8. PEG–TE–LiAC solution: 50% (w/v) polyethylene glycol 4000
(PEG 4000) in 1Â TE–LiAc solution (autoclave).
9. Dimethyl sulfoxide (DMSO).
2.7 Screening
of the Human PDZome
Library
1. SC -Leu plates.
2. Y2H PDZome array (frozen stock).
3. Wild-type and PBM mutant bait-containing AH109 strains.
4. SC -Leu liquid medium.
5. SC -Trp liquid medium.
6. YPD liquid medium.
7. SC -Leu, -Trp plates.
8. SC -Leu, -Trp, -His plates.
9. 96w polypropylene deep-well plates
10. 96-well U-bottom microplates.
Table 2
Amino acid dropout supplements and selection purposes
Selection purpose
Selective SC medium
Required supplement (8 mL/L)
Prey selection
SC -Leu
Trp, His, Ura
Bait selection
SC -Trp
Leu, His, Ura
Bait autoactivation readout
SC -Trp -His
Leu, Ura
Mating efficiency readout
-Leu -Trp
His, Ura
Y2H interaction readout
-Leu -Trp -His
Ura
Y2H Screening of PDZ Interactions
7