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Yeast cDNA Library Construction Technology Service

TekBiotech is committed to the technical development services of new drug antibodies and targeted peptide drugs. We have built a complete yeast surface display technology system. After years of project accumulation, combined with our experience in targeted peptide screening (cell screening, animal in vivo screening), we have specially launched the intracellular environment level interaction discovery verification system, namely the yeast hybridization verification system. To simplify, we combine the advantages of SMART technology and Gateway technology to provide customers with cDNA yeast library construction and its derived series of yeast hybridization verification services.

█ cDNA Display Library Construction Service

There are currently two cDNA library construction methods used: SMART technology and Gateway technology.

SMART technology was developed by Clontech in 1996. The full name of SMART technology is: Switching Mechanism At 5’ end of RNA Template, which is the abbreviation of RNA template 5’ end switching mechanism. It uses the template switching activity of reverse transcriptase to add specific sequences to the 5’ end of cDNA, thereby improving the efficiency of full-length cloning.

Gateway technology was developed by Invitrogen in 1999. Its characteristic is to introduce homologous recombination arms at both ends of the RNA template sequence, and then use homologous recombination (seamless cloning) to clone the vector. The convenience lies in replacing the construction vector of the target sequence and shuttling the vector between different host systems.

Both of the above methods can be used to construct high-quality cDNA display libraries separately. Tianjin Tech Biotech is good at using the SMART method combined with homologous recombination (homologous method of Gateway method) to construct cDNA display libraries. As shown in Figure 1, the SMART method synthesizes cDNA libraries:

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Figure 1 Schematic diagram of SMART method for synthesizing cDNA libraries

█ cDNA Display Library Type

According to customer requirements, TekBiotech can provide customers with uniform cDNA and non-uniform cDNA library construction services. Regardless of the type of library, customers can specify a specific vector (specific vectors require customers to provide blank plasmids and maps) for library construction. The types of cDNA libraries that TechBio can provide to customers are shown in Figure 2:

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█ Yeast cDNA Display Library Construction

TekBiotech provides customers with improved SMART cDNA library construction services. We obtain cDNA libraries using SMART technology, and perform homologous recombination of any vector by introducing homologous recombination arms at both ends of cDNA. This technology can be used to obtain prokaryotic cDNA libraries (please consult our scientists for more details) and eukaryotic yeast cDNA libraries. Figure 3 shows a yeast-type cDNA library constructed using SMART technology.

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Figure 3 Yeast display cDNA library based on improved SMART technology

█ Yeast cDNA Display Library Construction Sample Requirements

Customers can prepare samples according to our requirements, or directly consult our scientists for detailed communication. As shown in Table 1, sample preparation requirements are as follows:

Sample Type	Sample Requirements	Storage Conditions (℃)	Transportation Conditions
Cell samples	Cell number greater than 2*10^7	-80	Dry ice
Animal tissue	Samples need to be prepared according to our instructions	-80	Dry ice
Plant samples	Samples need to be prepared according to our instructions	-80	Dry ice
Total RNA	> 50 μg (3 bands clearly visible)	-80	Dry ice

█ Yeast cDNA Library Service Content and Cycle

Steps	Service Content	Cycle
Step1: mRNA Preparation	1) Total RNA extraction (bands clearly visible, concentration>0.4ug/ul); 2) mRNA purification; 3) Delivery: total RNA quality verification report, mRNA quality verification report;	1 Week
Step2: cDNA Library Construction Service	1) cDNA synthesis and adapter connection (three-frame library) + cDNA Normalization (optional); 2) Recombination into yeast vector pGADT7 and electroporation; 3) Electroporation of the obtained cDNA library into competent E. coli cells for cloning and amplification to obtain the primary library; 4) Primary library verification: single clone sequencing for normalization effect verification; 5) Delivery: -- Primary cDNA library, amplified library plasmid (>500ug); -- Library capacity >1*10^7 CFU/ml; -- Average insert fragment 0.8-1.5 kb (different projects); -- Experimental report, library capacity titer results, sequencing raw data;	4-5 Weeks
Step3: Yeast cDNA Library Construction Service (optional)	1) Primary library for yeast super competent preparation electroporation; 2) Library capacity identification + single clone sequencing; 3) Delivery: -- Original yeast cDNA library glycerol bacteria; -- Library capacity >110^6 CFU/ml, library titer>210^7 CFU/ml; -- Experimental report, library titer results, sequencing raw data;	3-5 Weeks

█ Yeast cDNA Library Construction Technology Platform Service Advantages

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The RNA sample volume required

is small, suitable for precious

samples

Homogenization, effective

enrichment of low-abundance

expressed genes

Large library capacity

and high diversity

Traceable experimental records: library

QC quality control standards, Chinese and

English experimental reports, original

experimental records

One-to-one personalized solution

customization, design the best

construction solution according to

customer needs

Yeast cDNA Library Construction Technology Service Frequently Asked Questions

What is the construction of yeast libraries? What does SMART technology mean?

For two decades, yeast display methods have become a common tool for the discovery, stability improvement and affinity maturation of various protein scaffolds for antigen recognition. Yeast display is particularly suitable for the selection of heterodimeric proteins, such as antibodies and T cell receptors (TCRs), because it allows the rapid creation of libraries through homologous recombination driven by gap repair and the subsequent construction of combinatorial libraries after mating of yeasts of opposite mating types. Yeast libraries are composed of many different gene clones that are able to replicate and express in yeast cells after insertion into a vector. SMART amplification technology Total RNA is extracted from the organism and cDNA synthesis is performed using the RNA template 5' end switching mechanism (SMART) technology. Subsequently, the double-stranded cDNA is amplified by long-distance PCR, purified in yeast strains and co-transformed with the vector. The quality parameters of the constructed library are evaluated to validate the constructed library.
What are the advantages of SMART technology? What are the general applications?

Even if the amount of the customer's initial sample RNA is small, SMART technology can complete the amplification of the sample's full-length cDNA with high quality and efficiency. This technology is more suitable for cloning low-abundance expression genes. After homogenization treatment by some technical means, we can find that SMART technology significantly reduces the redundancy of genes, helping us to effectively enrich those genes with low expression, which can better improve the gene diversity in the yeast library we constructed. The operation process of SMART technology is simpler and faster, which is conducive to scientific researchers to conduct experiments quickly in the laboratory. SMART technology uses the template switching activity of reverse transcriptase to add specific sequences to the 5' end of cDNA, which can improve the efficiency of full-length cloning. TechBio has a strict quality control process. Through the quality control process, the yeast library constructed by TechBio using SMART technology can meet high standards in terms of library capacity, average insert length and clone positive rate. TechBio's researchers can construct high-quality and reliable yeast libraries to provide strong support for customers' in-depth research in different life science fields.
In the yeast library construction service, what are the requirements of SMART technology for the RNA starting amount?

One of the significant advantages of SMART technology, which has been used in the field of molecular biology, is that it is highly tolerant of the RNA starting amount. In the experiment, this technology only requires a very low RNA starting amount, usually only a few micrograms (μg) of high-quality RNA, to start and successfully complete the complex library construction process. This feature is crucial for projects with precious samples and difficult to obtain RNA samples, such as our common rare cell type analysis, low-abundance gene expression research, or limited clinical sample resources in hospitals. If you encounter the above situation, SMART technology can greatly lower the threshold of the experiment and will not limit our experiment by the sample amount. Although the sample amount required by SMART technology is very low, we still have to pay attention to the quality of the RNA sample, which has a significant impact on the diversity of the final constructed library. Therefore, when using SMART technology, strict RNA extraction and purification methods must also be adopted.
How efficient is SMART technology in constructing yeast libraries? How to ensure the diversity of yeast libraries constructed by SMART technology? How do we solve the above problems?

SMART technology introduces a switching reaction of reverse transcriptase at the 5' end of RNA through its unique amplification mechanism. However, compared with some other library construction technologies, Gateway recombination technology, SMART technology may not be as efficient as other technologies in some aspects. In the face of this technical shortcoming, TechBio's researchers have actively explored and optimized the experimental conditions. The optimization of the experimental scheme includes but is not limited to adjusting the proportion of required solvents in the RNA extraction process and temperature control in the extraction, controlling the reaction time and number of cycles in the experiment, and optimizing the pH value in the reaction system. When we face the problem of insufficient library diversity, we find that the key to ensuring library diversity lies in the diversity and integrity of the starting RNA sample. During the extraction process, it is crucial to ensure its integrity, so we must prevent its degradation and contamination. As for the library construction stage, the deviation in the control process cannot be ignored, aiming to reduce the excessive phenomenon of specific sequences, thereby effectively improving the diversity of the yeast library.
In the yeast library construction service, how do we evaluate the quality of the yeast library constructed by SMART technology? What if the yeast library constructed by SMART technology has false positives? How do we solve it?

In the yeast library construction service, the quality of the yeast library constructed by SMART technology is directly related to the effectiveness and accuracy of subsequent research. The first is the library capacity, which can directly reflect the degree of genetic diversity contained in the library. High-capacity libraries can provide a wider range of gene resources for screening and research. Next is the size of the inserted fragment. The inserted fragment is related to the gene fragment in the library. At the same time, the number of positive clones in a library can also evaluate the quality. Positive clones can show the number of clones that correctly insert the target gene fragment. A high number of positive clones indicates a high screening and success rate. At the same time, false positive problems often occur in library construction and are not limited to SMART technology. In the face of false positives, in order to reduce false positives, we strictly control various experimental conditions, conduct multiple verifications and bioinformatics analysis on the results, etc.

What is the construction of yeast libraries? What does SMART technology mean?

For two decades, yeast display methods have become a common tool for the discovery, stability improvement and affinity maturation of various protein scaffolds for antigen recognition. Yeast display is particularly suitable for the selection of heterodimeric proteins, such as antibodies and T cell receptors (TCRs), because it allows the rapid creation of libraries through homologous recombination driven by gap repair and the subsequent construction of combinatorial libraries after mating of yeasts of opposite mating types. Yeast libraries are composed of many different gene clones that are able to replicate and express in yeast cells after insertion into a vector. SMART amplification technology Total RNA is extracted from the organism and cDNA synthesis is performed using the RNA template 5' end switching mechanism (SMART) technology. Subsequently, the double-stranded cDNA is amplified by long-distance PCR, purified in yeast strains and co-transformed with the vector. The quality parameters of the constructed library are evaluated to validate the constructed library.
What are the advantages of SMART technology? What are the general applications?

Even if the amount of the customer's initial sample RNA is small, SMART technology can complete the amplification of the sample's full-length cDNA with high quality and efficiency. This technology is more suitable for cloning low-abundance expression genes. After homogenization treatment by some technical means, we can find that SMART technology significantly reduces the redundancy of genes, helping us to effectively enrich those genes with low expression, which can better improve the gene diversity in the yeast library we constructed. The operation process of SMART technology is simpler and faster, which is conducive to scientific researchers to conduct experiments quickly in the laboratory. SMART technology uses the template switching activity of reverse transcriptase to add specific sequences to the 5' end of cDNA, which can improve the efficiency of full-length cloning. TechBio has a strict quality control process. Through the quality control process, the yeast library constructed by TechBio using SMART technology can meet high standards in terms of library capacity, average insert length and clone positive rate. TechBio's researchers can construct high-quality and reliable yeast libraries to provide strong support for customers' in-depth research in different life science fields.
In the yeast library construction service, what are the requirements of SMART technology for the RNA starting amount?

One of the significant advantages of SMART technology, which has been used in the field of molecular biology, is that it is highly tolerant of the RNA starting amount. In the experiment, this technology only requires a very low RNA starting amount, usually only a few micrograms (μg) of high-quality RNA, to start and successfully complete the complex library construction process. This feature is crucial for projects with precious samples and difficult to obtain RNA samples, such as our common rare cell type analysis, low-abundance gene expression research, or limited clinical sample resources in hospitals. If you encounter the above situation, SMART technology can greatly lower the threshold of the experiment and will not limit our experiment by the sample amount. Although the sample amount required by SMART technology is very low, we still have to pay attention to the quality of the RNA sample, which has a significant impact on the diversity of the final constructed library. Therefore, when using SMART technology, strict RNA extraction and purification methods must also be adopted.
How efficient is SMART technology in constructing yeast libraries? How to ensure the diversity of yeast libraries constructed by SMART technology? How do we solve the above problems?

SMART technology introduces a switching reaction of reverse transcriptase at the 5' end of RNA through its unique amplification mechanism. However, compared with some other library construction technologies, Gateway recombination technology, SMART technology may not be as efficient as other technologies in some aspects. In the face of this technical shortcoming, TechBio's researchers have actively explored and optimized the experimental conditions. The optimization of the experimental scheme includes but is not limited to adjusting the proportion of required solvents in the RNA extraction process and temperature control in the extraction, controlling the reaction time and number of cycles in the experiment, and optimizing the pH value in the reaction system. When we face the problem of insufficient library diversity, we find that the key to ensuring library diversity lies in the diversity and integrity of the starting RNA sample. During the extraction process, it is crucial to ensure its integrity, so we must prevent its degradation and contamination. As for the library construction stage, the deviation in the control process cannot be ignored, aiming to reduce the excessive phenomenon of specific sequences, thereby effectively improving the diversity of the yeast library.
In the yeast library construction service, how do we evaluate the quality of the yeast library constructed by SMART technology? What if the yeast library constructed by SMART technology has false positives? How do we solve it?

In the yeast library construction service, the quality of the yeast library constructed by SMART technology is directly related to the effectiveness and accuracy of subsequent research. The first is the library capacity, which can directly reflect the degree of genetic diversity contained in the library. High-capacity libraries can provide a wider range of gene resources for screening and research. Next is the size of the inserted fragment. The inserted fragment is related to the gene fragment in the library. At the same time, the number of positive clones in a library can also evaluate the quality. Positive clones can show the number of clones that correctly insert the target gene fragment. A high number of positive clones indicates a high screening and success rate. At the same time, false positive problems often occur in library construction and are not limited to SMART technology. In the face of false positives, in order to reduce false positives, we strictly control various experimental conditions, conduct multiple verifications and bioinformatics analysis on the results, etc.

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Yeast cDNA Library Construction Technology Service Frequently Asked Questions

What is the construction of yeast libraries? What does SMART technology mean?

What are the advantages of SMART technology? What are the general applications?

In the yeast library construction service, what are the requirements of SMART technology for the RNA starting amount?