Membrane System Y2H Library Construction Service with Invitrogen-Gateway Method

The traditional yeast two-hybrid can only detect the interactions between proteins that occur in the nucleus, but cannot detect the interactions between membrane proteins; Many proteins need to undergo post-transcriptional modification in the endoplasmic reticulum or cytoplasm to interact with other proteins, which cannot be detected by traditional yeast two-hybrid; Some proteins themselves are transcriptional activators, which can regulate the expression of reporter genes, which cannot be detected by traditional yeast two-hybrid. The DUAL-membrane membrane protein yeast two-hybrid system uses DUAL-membrane technology to break through the limitations of traditional yeast two-hybrid and expand the application range of yeast two-hybrid. Based on the traditional yeast two-hybrid system, it cleverly uses the split-ubiquitin system to screen protein interactions.

Gateway system uses site-specific recombination technology (Cloneminer cDNA library construction kit) to construct a library. It reverse-transcribes mRNA into cDNA to construct a library, without PCR, to maximize the quality and fidelity of the library. Using gateway's high-efficiency fragment and vector recombination ligation method has high ligation efficiency, which can greatly increase the original library capacity (original library capacity above 1*10⁷ CFU).

Service introduction

Creative BioMart combines years of library construction experience to provide yeast two-hybrid library construction services based on the Gateway method in membrane system. Relying on the advantages of invitrogen's gateway method library construction system, it continues to optimize conditions to achieve a higher level of yeast nucleoprotein two-hybrid library construction.

Experiment workflow

• RNA extraction and mRNA isolation
• Enzymatic synthesis of cDNA (synthesize directly using enzymatic method without undergoing PCR amplification process)
• Connect adapters to cDNA and separate cDNA by length
• Recombination of cDNA and membrane protein yeast two-hybrid library vector (pPR3N / pDEST22) --- Gateway homologous recombination method
• Electrotransformation of recombinant products
• Library detection and plasmid extraction --- invitrogen method uses liquid culture for library amplification and extraction

Material requirements

• Total RNA > 300 μg
• Animal sample > 1 g
• Plant sample > 2 g
• Cell sample > 1*10⁷

Delivery result

• Yeast two-hybrid library of glycerol bacteria (storage volume > 1*10⁷CFU, average insert fragment > 1200 bp, positive rate > 95%)
• Yeast two-hybrid library plasmid
• Library construction report (electronic version)
• Library construction picture (JPG)
• (Optional) Library glycerol bacteria (transformed into yeast cells, storage volume > 1*10⁸ CFU/ml).

Project cycle

It takes about 45 working days to complete membrane system yeast two-hybrid library construction process based on invitrogen-gateway method.

Service process

Please let us know the details of your yeast related projects through online consultation. We will reply to you in time and provide you with a comprehensive evaluation report on your project, including the feasibility evaluation of the project and the detailed experimental plan designed by our professionals. As our condition optimization is completed, the result report and other final products will be delivered to you immediately.

Creative BioMart has accumulated many years of experience in the field of yeast research, helping our customers accelerate research in all fields using yeast as a research tool, and improve the overall success rate of the project. Please tell us your project requirements, and we will provide you with a full service from strategy design to final report. If you have any questions, please feel free to contact us.