Membrane System Y2H Library Construction Service with Clontech-SMART 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. Its main feature is that it can be used to study the interaction between membrane protein-membrane protein and membrane protein-soluble protein. It can be used for the screening of expression libraries, and it can also be used to verify the interaction between two known proteins (one of which is a membrane protein).

Yeast two-hybrid library construction process of clontech SMART system: 1. Using total RNA as template, reverse transcribed into cDNA by SMART method, the amount of materials is small; 2. Using high temperature resistant (60℃) reverse transcriptase, it can open more mRNA High-grade hairpin structure, the proportion of full length obtained is relatively high; 3. Construction of a library can be used for yeast single-, two-, and three-hybrid; 4. Library index is high.The library capacity of the original library is greater than 1×10⁷ CFU, and the average inserted fragment is greater than 1000bp, the positive clone rate is greater than 95%.

Service introduction

Creative BioMart combines years of library construction experience to provide yeast two-hybrid library construction services in membrane system based on the clontech-SMART method. Relying on the advantages of clontech's SMART 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
• cDNA synthesis (PCR amplification)
• Recombination of cDNA and membrane protein yeast two-hybrid library vector (pPR3N / pGADT7) --- (SMART method --- restriction digestion and ligation)
• Transform the recombinant products into yeast
• Library detection and plasmid extraction

Material requirements

• Total RNA > 20 μg
• Animal sample > 0.5 g
• Plant sample > 1 g
• Cell sample > 1*10⁷

Delivery result

• Yeast two-hybrid library of glycerol bacteria (storage volume > 1*10⁷ CFU, 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 35 working days to complete membrane system yeast two-hybrid library construction process based on clontech-SMART 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.