Matchmaker Insert Check PCR Mix 1

Use colony PCR to quickly analyze your bait-specific reporter strains and sort your positive candidate clones

Use colony PCR to quickly analyze your bait-specific reporter strains and sort your positive candidate clones. Panel A: Colony PCR was performed using Matchmaker Insert Check PCR Mix 1 to analyze the insertion sites of Y1HGold pAbAi integrant strains, which contain the pAbAi reporter vector with or without a bait sequence (i.e., three tandem repeats of either the Oct4 or p53 binding site). Lane 1: Y1HGold. Lane 2: Y1HGold [AbAi]. Lane 3: Y1HGold [3xOct4-AbAi]. Lane 4: Y1HGold [3xp53-AbAi]. Lane M: 1 kb ladder. The strain lacking pAbAi produced no PCR product, while those containing pAbAi integrants generated amplicons of approximately 1.4 kb. Panel B: Matchmaker Insert Check PCR Mix 2 was used to amplify prey vector inserts from 15 randomly selected positive colonies obtained using a Matchmaker Gold Screening System (Lanes 3-17). The results allowed the clones to be quickly sorted for further analysis. Lane 1: No template control. Lanes M and M2: 1 kb ladder. Lane M1: 100 bp ladder.

Use SMART technology and yeast biology to construct and screen your library

Use SMART technology and yeast biology to construct and screen your library. Your library is simultaneously constructed and screened directly in yeast. First, SMART cDNA synthesis technology is used to create a pool of cDNA that is flanked by sequence that is homologous to that at the ends of the linearized prey vector, pGADT7-Rec. Next, the newly created Y1HGold-Bait reporter strain is cotransformed with the cDNA pool and pGADT7-Rec, which undergo homologous recombination within the yeast. The yeast cells are then plated on SD/-Leu/+AbA to select for colonies that have an active reporter (i.e., positive Y1H interactions)

Screening for protein-DNA interactions with the Matchmaker Gold One-Hybrid System

Screening for protein-DNA interactions with the Matchmaker Gold One-Hybrid System. One to three copies of the DNA target sequence are cloned into the pAbAi reporter vector, which is then integrated into the Y1HGold genome to create a bait-specific reporter strain. Activation of the AbA resistance gene occurs if a prey protein from the library binds to the bait sequence.