6. Resuspend the cells in 15 mL of 1Â TE–LiAc.
7. Centrifuge the cell suspension as described in step 3.
8. Resuspend the cell pellet in 1 mL of 1Â TE–LiAc by pipetting
up and down.
9. In two sterile 1.5 mL microtubes, distribute in each tube 5 μL
of ssDNA salmon sperm carrier DNA, 100 ng of either the
wild-type or mutant (ΔPBM) bait DNA plasmids, 30 μL of
AH109 cell suspension from step 8 and 100 μL of freshly
prepared PEG–TE–LiAc solution (see Note 1).
10. Homogenize by vortexing thoroughly the cell suspension and
incubate for at least 1 h at 30
C.
11. Add DMSO to a 10% final concentration to each tube (see Note
2) and give a 15 min heat shock to the cells by placing them in a
42
C water bath.
12. Spin down the cells at 800 Â g for 8 min and discard the
supernatant.
13. Resuspend the cells in 150 μL of sterile ddiH 2 O.
14. Plate 50 μL of the cell suspension on Agar SC-Trp plates.
15. Incubate the plates for 2–3 days at 30
C until colonies appear.
Plates can be stored at 4
C at this stage.
3.5 Validating
the Bait Constructs
Before screening for potential PDZ partners, we strongly recommend to first check that none of the GAL4BD-bait fusions can
autoactivate the transcription of the GAL1-HIS3 reporter gene by
itself. This can be assessed by either of the two methods outlined
below.
1. A simple and straightforward test consist in streaking
GAL4BD-bait-containing AH109 Trp
+ transformants directly
on SC -Trp -His medium and monitoring cell growth after for
2–3 days of incubation at 30
C. Autoactivation of the His3
reporter gene by the GAL4BD-bait fusion will result in good
growth of all of the cells in the streak. In case of ambiguous
growth results, AH109 cells transformed with an empty
pGBT9 vector may prove useful as a convenient negative
control.
2. An alternative and slightly more rigorous method is to assess
the autoactivation capacity of a bait under the same cellular and
growth conditions as those used for the screening of the library,
that is, by mating the Trp
+ transformants that contain the bait
to be tested to Y187 cells transformed with the empty pACT2
vector.
3. An easy way to proceed with the mating step is to mix the two
mating partners with sterile tooth-picks in 250 μL of complete
YPD medium in a sterile 1.5 mL microtube.
10
Monica Castro-Cruz et al.
7. Centrifuge the cell suspension as described in step 3.
8. Resuspend the cell pellet in 1 mL of 1Â TE–LiAc by pipetting
up and down.
9. In two sterile 1.5 mL microtubes, distribute in each tube 5 μL
of ssDNA salmon sperm carrier DNA, 100 ng of either the
wild-type or mutant (ΔPBM) bait DNA plasmids, 30 μL of
AH109 cell suspension from step 8 and 100 μL of freshly
prepared PEG–TE–LiAc solution (see Note 1).
10. Homogenize by vortexing thoroughly the cell suspension and
incubate for at least 1 h at 30
C.
11. Add DMSO to a 10% final concentration to each tube (see Note
2) and give a 15 min heat shock to the cells by placing them in a
42
C water bath.
12. Spin down the cells at 800 Â g for 8 min and discard the
supernatant.
13. Resuspend the cells in 150 μL of sterile ddiH 2 O.
14. Plate 50 μL of the cell suspension on Agar SC-Trp plates.
15. Incubate the plates for 2–3 days at 30
C until colonies appear.
Plates can be stored at 4
C at this stage.
3.5 Validating
the Bait Constructs
Before screening for potential PDZ partners, we strongly recommend to first check that none of the GAL4BD-bait fusions can
autoactivate the transcription of the GAL1-HIS3 reporter gene by
itself. This can be assessed by either of the two methods outlined
below.
1. A simple and straightforward test consist in streaking
GAL4BD-bait-containing AH109 Trp
+ transformants directly
on SC -Trp -His medium and monitoring cell growth after for
2–3 days of incubation at 30
C. Autoactivation of the His3
reporter gene by the GAL4BD-bait fusion will result in good
growth of all of the cells in the streak. In case of ambiguous
growth results, AH109 cells transformed with an empty
pGBT9 vector may prove useful as a convenient negative
control.
2. An alternative and slightly more rigorous method is to assess
the autoactivation capacity of a bait under the same cellular and
growth conditions as those used for the screening of the library,
that is, by mating the Trp
+ transformants that contain the bait
to be tested to Y187 cells transformed with the empty pACT2
vector.
3. An easy way to proceed with the mating step is to mix the two
mating partners with sterile tooth-picks in 250 μL of complete
YPD medium in a sterile 1.5 mL microtube.
10
Monica Castro-Cruz et al.