6. Check for the presence of bait DNA fragments among the
purified recombinant pDONr/pZeo
® clones. This can be easily
assessed through a BsrGI restriction analysis as both attB1 and
attB2 sites harbor a BsrGI restriction site. Assuming that the
bait DNA sequence itself contains no BsrGI site, restriction
with this enzyme should release DNA fragments with a size
close to that of the amplified bait products.
7. In addition to the restriction analysis it is strongly recommended to verify the DNA sequence of a few bait-containing
pDONr/pZeo
® entry clones by performing a DNA sequencing with M13-For and/or M13-Rev primers.
3.3 Cloning
of the Bait Constructs
in pGBT9-GW
This protocol explains how to transfer bait DNA fragments
between pDONr/pZeo
® entry clones and pGBT9-GW, the destination Y2H vector in which the bait genes will be fused downstream of the GAL4-DB domain.
1. Mix 300 ng of the relevant Bait-pDONr/pZeo
® entry clone
with 250 ng of pGBT9-GW. Add 2 μL of TE buffer and 2 μL of
the LR recombinase and let the reaction incubate for at least
1 h on the bench.
2. Transform the recombination mix in chemically competent
bacteria and select transformants by plating bacterial cells on
1Â ampicillin-supplemented LB-Agar plates for 16 h at 37
C.
3. Inoculate a few colonies in 3 mL of 1Â ampicillinsupplemented liquid LB and grow them overnight at 37
C.
4. Proceed with the purification of the plasmid DNA and with the
restriction analysis as described in Subheading 3.3 (steps 5 and
6) to identify bait-containing pGBT9-GW clones.
3.4 Yeast
Transformation
of the Bait Constructs
The procedure to transform pGBT9-GW vectors harboring the
wild-type (WT) or mutant (ΔPBM) bait constructs in the AH109
yeast strain is explained below.
1. Inoculate a colony of AH109 yeast cells in 3 mL of YPD liquid
medium and incubate overnight at 30
C under vigorous
orbital agitation.
2. The next day use the preculture to inoculate 30 mL of YPD
liquid medium at a 1:100 (v/v) dilution.
3. When the culture reaches an OD 600 of 0.6 to 0.8, spin down
the cells at 800 Â g for 8 min, discard the supernatant, and
resuspend the cell pellet in 30 mL of sterile ddH 2 O by pipetting up and down.
4. Spin down the cells as indicated in step 3.
5. Prepare 50 mL of 1Â TE–LiAc solution using the 10Â TE
buffer and 10Â LiAC stock solutions.
Y2H Screening of PDZ Interactions
9
purified recombinant pDONr/pZeo
® clones. This can be easily
assessed through a BsrGI restriction analysis as both attB1 and
attB2 sites harbor a BsrGI restriction site. Assuming that the
bait DNA sequence itself contains no BsrGI site, restriction
with this enzyme should release DNA fragments with a size
close to that of the amplified bait products.
7. In addition to the restriction analysis it is strongly recommended to verify the DNA sequence of a few bait-containing
pDONr/pZeo
® entry clones by performing a DNA sequencing with M13-For and/or M13-Rev primers.
3.3 Cloning
of the Bait Constructs
in pGBT9-GW
This protocol explains how to transfer bait DNA fragments
between pDONr/pZeo
® entry clones and pGBT9-GW, the destination Y2H vector in which the bait genes will be fused downstream of the GAL4-DB domain.
1. Mix 300 ng of the relevant Bait-pDONr/pZeo
® entry clone
with 250 ng of pGBT9-GW. Add 2 μL of TE buffer and 2 μL of
the LR recombinase and let the reaction incubate for at least
1 h on the bench.
2. Transform the recombination mix in chemically competent
bacteria and select transformants by plating bacterial cells on
1Â ampicillin-supplemented LB-Agar plates for 16 h at 37
C.
3. Inoculate a few colonies in 3 mL of 1Â ampicillinsupplemented liquid LB and grow them overnight at 37
C.
4. Proceed with the purification of the plasmid DNA and with the
restriction analysis as described in Subheading 3.3 (steps 5 and
6) to identify bait-containing pGBT9-GW clones.
3.4 Yeast
Transformation
of the Bait Constructs
The procedure to transform pGBT9-GW vectors harboring the
wild-type (WT) or mutant (ΔPBM) bait constructs in the AH109
yeast strain is explained below.
1. Inoculate a colony of AH109 yeast cells in 3 mL of YPD liquid
medium and incubate overnight at 30
C under vigorous
orbital agitation.
2. The next day use the preculture to inoculate 30 mL of YPD
liquid medium at a 1:100 (v/v) dilution.
3. When the culture reaches an OD 600 of 0.6 to 0.8, spin down
the cells at 800 Â g for 8 min, discard the supernatant, and
resuspend the cell pellet in 30 mL of sterile ddH 2 O by pipetting up and down.
4. Spin down the cells as indicated in step 3.
5. Prepare 50 mL of 1Â TE–LiAc solution using the 10Â TE
buffer and 10Â LiAC stock solutions.
Y2H Screening of PDZ Interactions
9