9. Duplicate the library by transferring 30 μL of each of the PDZ
clones from step 5 in a YPD-containing well of the 96w plates
prepared in step 7.
10. Tightly seal the plates with an adhesive aluminum foil and let
the cells mate for at least 5 h at 30
C under gentle (140 rpm)
agitation.
11. Eliminate the medium by spinning down the cells for 10 min at
800 Â g in a centrifuge equipped with swinging plate carriers.
Using a multichannel pipette, wash the cell pellets once with
200 μL of sterile ddH 2 O before resuspending them in 20 μL of
sterile ddH 2 O.
12. For each PDZ clone, spot 5 μL of the mating cell suspension
on SC -Leu, -Trp and 8 μL on SC -Leu, -Trp, -His plates.
13. Incubate all SC Agar plates at 30
C until colony growth can be
assessed on the different media (Fig. 3). Check mating efficiency by monitoring growth on SC -Leu, -Trp medium 2 to
3 days after plating. Growth on SC -Leu, -Trp, -His medium
usually takes 2 to 3 more days and may vary to a large degree
depending on the strength of the Y2H interaction (see Note 7).
14. Scan all the plates and score the positive clones that emerged
on the readout conditions, that is, SC -Leu, -Trp, -His agar
plates, with the WT bait and not with the ΔPBM mutant (see
Notes 8 and 9).
15. Duplicate the screening experiment in the same conditions.
4 Notes
1. Because the PEG–TE–LiAc solution is quite viscous, cutting
the end of a tip with a sterile razor blade greatly helps pipetting
of this solution. Also try to go slowly when giving several
rounds of up and down pipetting to homogenize the cells in
this solution.
2. DMSO is used to enhance DNA entry but can prove quite toxic
to the cells and may trigger cell death, in which case we recommend to avoid including this reagent during the heat shock
procedure.
3. When manipulating the whole Y2H PDZome array, the use of a
multichannel pipette is strongly recommended as it greatly
facilitates handling and helps maintaining the 96-well plate
format of the array. Operating this way will also greatly contribute to reducing mistakes in the identification of positive
clones.
Y2H Screening of PDZ Interactions
13
clones from step 5 in a YPD-containing well of the 96w plates
prepared in step 7.
10. Tightly seal the plates with an adhesive aluminum foil and let
the cells mate for at least 5 h at 30
C under gentle (140 rpm)
agitation.
11. Eliminate the medium by spinning down the cells for 10 min at
800 Â g in a centrifuge equipped with swinging plate carriers.
Using a multichannel pipette, wash the cell pellets once with
200 μL of sterile ddH 2 O before resuspending them in 20 μL of
sterile ddH 2 O.
12. For each PDZ clone, spot 5 μL of the mating cell suspension
on SC -Leu, -Trp and 8 μL on SC -Leu, -Trp, -His plates.
13. Incubate all SC Agar plates at 30
C until colony growth can be
assessed on the different media (Fig. 3). Check mating efficiency by monitoring growth on SC -Leu, -Trp medium 2 to
3 days after plating. Growth on SC -Leu, -Trp, -His medium
usually takes 2 to 3 more days and may vary to a large degree
depending on the strength of the Y2H interaction (see Note 7).
14. Scan all the plates and score the positive clones that emerged
on the readout conditions, that is, SC -Leu, -Trp, -His agar
plates, with the WT bait and not with the ΔPBM mutant (see
Notes 8 and 9).
15. Duplicate the screening experiment in the same conditions.
4 Notes
1. Because the PEG–TE–LiAc solution is quite viscous, cutting
the end of a tip with a sterile razor blade greatly helps pipetting
of this solution. Also try to go slowly when giving several
rounds of up and down pipetting to homogenize the cells in
this solution.
2. DMSO is used to enhance DNA entry but can prove quite toxic
to the cells and may trigger cell death, in which case we recommend to avoid including this reagent during the heat shock
procedure.
3. When manipulating the whole Y2H PDZome array, the use of a
multichannel pipette is strongly recommended as it greatly
facilitates handling and helps maintaining the 96-well plate
format of the array. Operating this way will also greatly contribute to reducing mistakes in the identification of positive
clones.
Y2H Screening of PDZ Interactions
13