A simple and highly efficient system for the simultaneous construction and screening of a cDNA library for protein-DNA interactions. SMART cDNA is synthesized from your RNA sample and then used to construct a library directly in a Y1HGold yeast reporter strain containing your DNA sequence of interest. Positive protein-DNA interactions from the library convey resistance to the yeast antibiotic, Aureobasidin A (AbA).
Overview
- Highest-performing yeast one-hybrid system
- Aureobasidin A selection eliminates screening background
- Construct and screen SMART cDNA libraries directly in yeast
Applications
- Yeast one-hybrid screening
Get results fast!
With the Matchmaker Gold system, one-hybrid library screening is straightforward, quick, and easy:
Step 1: Create a bait construct by cloning 1–3 tandem repeats of the target DNA-binding sequence into pAbAi.
Step 2: Create a bait-specific reporter strain by transforming and integrating the linearized pBait-AbAi construct into the Y1H Gold yeast strain and selecting on SD/–Ura agar medium.
Step 3: Confirm the integration of the bait sequence using colony PCR and the Matchmaker Insert Check PCR Mix 1.
Step 4: Use SMART technology to synthesize cDNA containing ends that are homologous to the ends of the linearized pGADT7-Rec vector.
Step 5: Create and screen your library in a single step by cotransforming your bait-specific reporter strain with the SMART-generated cDNA and the linearized pGADT7-Rec vector, and plating on AbA-containing selective medium.
Step 6: Harvest the resulting colonies, which contain putative DNA-binding prey proteins, and analyze your library inserts using the Matchmaker Insert Check PCR Mix 2.
