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Yeast Three-hybrid Library Construction to study RNA-protein interactions (CAT#: STEM-MB-0018-WXH)

Introduction

Yeast three-hybrid system is a derivative of yeast two-hybrid (Y2H). It’s a kind of powerful tool to dissert RNA-protein interactions of interest and that typically consists of three chimeric components.<br />The first hybrid protein is made up of an RNA binding protein (RBD) fused to a DNA binding domain (DBD). The second fusion protein molecule contains a second RNA binding protein fused to the transcriptional activation domain (AD). The third hybrid part is an RNA molecule which bridges above two fusion proteins by providing two specific RNA targets for the RNA binding proteins. When this tripartite constituent forms at a promoter, the reporter gene is turned on, even transiently. And the expressed reporter products can be recognized by simple biochemical or phenotypic assays.




Applications

• Study RNA-protein interactions of interest
• Study of the interaction between two proteins and a third protein
• Experimental validation at the intracellular nucleic acid level

Procedure

1. Total RNA Extraction
2. mRNA isolation
3. Synthesis of cDNA
4. Connection of cDNA and linker
5. cDNA separation by length
6. Recombination of cDNA and yeast three-hybrid library vector
7. Electrotransformation of recombinant products
8. Library detection and plasmid extraction

Notes

Customer provides tissue, cells or total RNA