TEL:415-424-8222 or 800-662-CLON FAX:415-424-1064 or 800-424-1350 Technical Service Protocol # PT1031-1 Version # PR71132 page 11 CLONTECH Laboratories, Inc. V. Preparing Your Target-Reporter Constructs PLEASE READ THE ENTIRE PROTOCOL BEFORE BEGINNING. A. Target and Reporter Vector Information To use the MATCHMAKER One-Hybrid System to screen a Clontech Laboratories, Inc. clontech.com Protocol No. PT3000-1 4 Version No. PR7Y2399 SMART TM cDNA Library Construction Kit User Manual The SMART TM cDNA Library Construction Kit provides a method for produc-ing high-quality, full-length cDNA libraries from nanograms of total or poly A+ RNA. This kit contains two separate protocols All groups and messages Yeast two-hybrid assay Prey and bait vectors were co-transformed into the yeast strain AH109 using Yeast maker Yeast transformation system 2 according to the manufacturer's instructions (Clontech). The transformants were then grown on SD/-Trp/-Leu medium at 30°C for 5 days. The Matchmaker 3 Two-Hybrid system from Clontech, a Takara Bio Company, offers an improved approach to two-hybrid screening that reduces false positives and facilitates downstream analyses. Two-hybrid screening is a method of detecting protein-protein interactions that relies on transcriptional activation and reporter genes in yeast. provided with Clontech Matchmaker® Gold Yeast One-Hybrid Library Screening System. With the preparation of these vectors, the analyses can be separated into two parts: 1. Established the bait-reporter strains and test the minimal inhibitory concentration of aureobasidin A for the bait used in Y1H assays. 2. Clontech Laboratories, Inc. clontech.com Protocol No. PT3249-5 2 Version No. PR732196 Location of features: • Full-length S. cerevisiae ADH1 promoter (P ADH1): 7-1479 • GAL4 AD polypeptide with SV40 Nuclear Localization Signal (NLS) NLS: 1501-1557 GAL4 amino acids 768-881: 1561-1899 • T7 RNA polymerase promoter: 1905-1927 The yeast two-hybrid (Y2H) assay was performed using the Matchmaker™ Gold Yeast Two-Hybrid system (Clontech) and all protocols were adapted from the Clontech Yeast Protocols Handbook and the Matchmaker™ Gold Yeast Two-Hybrid user manual. To determine the autoactivation of candidate bait constructs, Y2HGold cells were transformed with the If so, then you can screen a yeast two-hybrid library. Clontech's Mate & Plate Libraries offer the simplest, most straight-forward method of screening a yeast two-hybrid library for interacting protein pairs. To screen these ready-to-go cDNA libraries simply: 1. Set up a mixed culture of your bait-expressing reporter strain Two yeast reporter strains, YM4271[p53HIS] and YM4271 [p53BLUE], were created according to the Matchmaker One-hybrid System User Manual (Clontech, Palo Alto, CA, USA) for reporter assay analysis in the MY1H. These two strains contain three tandem copies of the consensus p53 binding site upstream of the HIS3 and lacZ reporter genes, respectively.