bait gene. The codon sequence shown here within the attB1
site is in frame with the coding sequence of the upstream
GAL4BD region. It is therefore critical to make sure that the
sequence of the bait is in frame with the indicated attB1 reading
sequence as it is the case here with the TNN fusion codon.
2. Design a Rev-attB2-ORFwt reverse primer based on the
sequence 5
0 -GGGG AC CAC TTT GTA CAA GAA AGC
TGG GTC TTA NNN-3
0 where the attB2 recombination site
is underlined and N letters correspond to the reverse and
complementary 3
0 -end wild-type sequence of the bait construct. Pay attention to the fact that a stop codon has to be
placed between the 3
0 -end of the target gene sequence and the
attB2 site as additional amino acid residues at the C-terminus
of the bait are likely to disrupt its PBM properties. In the above
Rev-attB2-ORFwt sequence, a TAA stop codon (denoted in
bold letters) has been introduced but the endogenous stop
codon of the bait target can be used as well.
3. Following the same guidelines as described in item 2, design a
second Rev-attB2-ORFmut reverse primer in which the
sequence of the last three C-terminal codons of the bait is
lacking. This primer will serve for the cloning of the PBM
mutant bait construct.
2.2 PCR
Amplification
and Purification
of the Bait Constructs
1. 5 mM dNTPs.
2. 10 μM forward and reverse primers.
3. Bait DNA to be used as a template for the PCR reaction.
4. High-fidelity DNA polymerase.
5. Concentrated stocks of reaction buffer and magnesium chloride provided with the DNA polymerase.
6. TAE buffer: 40 mM Tris, 40 mM acetate, 1 mM EDTA,
pH 8.3.
7. 1% or 2% agarose gels prepared in TAE buffer.
8. Gel DNA extraction kit of your choice. Be aware that some kits
may not be optimized for the recovery of small DNA
fragments.
2.3 Cloning
of the Bait Constructs
1. pDONr/pZeo
® plasmid DNA.
2. pGBT9-GW plasmid DNA.
3. BP recombinase
® .
4. LR recombinase
®
.
5. TE buffer: 10 mM Tris–HCl, pH 8; and 1 mM EDTA, pH 8.
6. Chemically competent DH5α bacteria.
Y2H Screening of PDZ Interactions
5
site is in frame with the coding sequence of the upstream
GAL4BD region. It is therefore critical to make sure that the
sequence of the bait is in frame with the indicated attB1 reading
sequence as it is the case here with the TNN fusion codon.
2. Design a Rev-attB2-ORFwt reverse primer based on the
sequence 5
0 -GGGG AC CAC TTT GTA CAA GAA AGC
TGG GTC TTA NNN-3
0 where the attB2 recombination site
is underlined and N letters correspond to the reverse and
complementary 3
0 -end wild-type sequence of the bait construct. Pay attention to the fact that a stop codon has to be
placed between the 3
0 -end of the target gene sequence and the
attB2 site as additional amino acid residues at the C-terminus
of the bait are likely to disrupt its PBM properties. In the above
Rev-attB2-ORFwt sequence, a TAA stop codon (denoted in
bold letters) has been introduced but the endogenous stop
codon of the bait target can be used as well.
3. Following the same guidelines as described in item 2, design a
second Rev-attB2-ORFmut reverse primer in which the
sequence of the last three C-terminal codons of the bait is
lacking. This primer will serve for the cloning of the PBM
mutant bait construct.
2.2 PCR
Amplification
and Purification
of the Bait Constructs
1. 5 mM dNTPs.
2. 10 μM forward and reverse primers.
3. Bait DNA to be used as a template for the PCR reaction.
4. High-fidelity DNA polymerase.
5. Concentrated stocks of reaction buffer and magnesium chloride provided with the DNA polymerase.
6. TAE buffer: 40 mM Tris, 40 mM acetate, 1 mM EDTA,
pH 8.3.
7. 1% or 2% agarose gels prepared in TAE buffer.
8. Gel DNA extraction kit of your choice. Be aware that some kits
may not be optimized for the recovery of small DNA
fragments.
2.3 Cloning
of the Bait Constructs
1. pDONr/pZeo
® plasmid DNA.
2. pGBT9-GW plasmid DNA.
3. BP recombinase
® .
4. LR recombinase
®
.
5. TE buffer: 10 mM Tris–HCl, pH 8; and 1 mM EDTA, pH 8.
6. Chemically competent DH5α bacteria.
Y2H Screening of PDZ Interactions
5