recombination sites flanking the target gene sequence. Introduction of these sites is easily achieved by PCR using oligonucleotides
designed as follows.
1. Design a For-attB1-ORF forward primer based on the
sequence 5
0 -GGGG ACA AGT TTG TAC AAA AAA GCA
GGC TNN NNN-3
0 where the attB1 recombination site is
underlined and N letters correspond to the 5
0 -end of your
Fig. 2 Construction of the hPDZome array for yeast two-hybrid screening. (a) The human PDZome was built
using the Gateway
®
system. The 266 entry clones in the pZeo vector were provided in 96-well plates by
NZYTech. Each entry clone corresponds to one ORF of the hPDZome. (b) The 266 PDZ ORFs were subcloned
into the pACT2-AD vector using Gateway
®
LR clonase and verified by sequencing. (c) Once validated, each
pACT2-AD PDZ prey was transformed into the Y187 prey yeast strain and transformants were selected by
plating on synthetic complete agar plates without leucine (SC agar -Leu). The final array ready for mating
consists of three 96-well plates with yeast clones expressing the hPDZome fused to the Gal4 activation
domain (AD)
4
Monica Castro-Cruz et al.