"Since Field et al. established the first yeast-based intracellular system for detecting protein interactions in 1989, the yeast two-hybrid system has been increasingly used to identify new proteins and proteins. Interactions, identification of protein cascade substrates, and identification of the effects of mutations on protein-protein binding, etc. According to literature statistics, at least half of the currently known protein interactions were discovered through yeast two-hybrid experiments.” And the library screening results are direct! Determined by the quality of the library, constructing a high-quality library is a key factor in obtaining good screening results.
We have 13 years of experience in library construction and have completed the construction of thousands of various libraries. The technologies and kits used are the best quality library construction reagents on the market, ensuring that every library constructed is All indicators of a library are of the highest standards in the market, and our success rate in constructing libraries is over 98%. Regarding our library construction products, we promise the following indicators. Each indicator is the highest among domestic companies.
Shanghai Baoji combines many years of library construction experience to launch the "All-Direct" library construction technology. Our products are superior to other construction methods (mainly Takara's SAMRT method and Invitrogen's gateway method). It has great advantages in terms of quality and quality.
Regarding our library construction products, we promise the following indicators, each of which is the highest among domestic companies:
The original library capacity is greater than 1*10^7CFU ;
The average inserted fragment is greater than 1200bp;
The clone positive rate is greater than 95.
? 1. We use homologous recombination to construct the library without any endonuclease cutting and ligation processes to ensure that the gene will not be cut by enzymes and ensure the integrity of the gene. properties, while Clonetech's is constructed using enzyme cleavage ligation.
? 2. Since we use recombination to recombine cDNA into the vector, it is much more efficient than clonetech's SMART T4 ligase method. We promise that the library capacity of the original library is within 1 *10^7 or more. Moreover, the recombination method has very low false positives. Some of our customers have tested 30,000 clones at a time, and not a single one has become empty. We promise that the positive rate is greater than 98%, which is far beyond the reach of enzyme digestion and ligation methods.
? 3. Clonetech’s SMART method uses PCR to synthesize the second strand. Due to the selective inhibition and competitive amplification of PCR, this will result in very high gene redundancy and low abundance. High-density genes cannot be amplified by PCR and will be lost, and long fragments of genes will also be lost. In addition, there will be a lot of repeated sequences, especially when the starting RNA sample is small (for example, 15 μg RNA or less is required) More than one cycle of PCR amplification will greatly reduce the quality of the library), and greatly reduce the number of genes contained in the library. However, our cDNA synthesis method uses a variety of enzymes to directly synthesize the second strand at a constant temperature of 16 degrees, so that It is completely loyal to the genetic condition of the sample itself and will not artificially change the genetic condition and gene size in the sample, which greatly improves the quality of the library.
4. We use a high-quality reverse transcriptase, which is recognized as the best reverse transcriptase and has better reverse transcription properties than clonetech's reverse transcriptase. High temperature (55 degrees Celsius reversal, clonetech's reverse transcriptase uses 42 degrees reversal, higher temperature can open the secondary structure of RNA and obtain longer cDNA), reverse transcription efficiency and cDNA length are better many. We promise that the average insert fragment of the library is more than 1.3Kbp, and the minimum length is also more than 1Kbp. However, the libraries constructed by clonetech's SMART method are generally only a few hundred BP.
Library construction process:
1. RNA extraction
2. mRNA extraction
3. Enzymatic synthesis of cDNA
p>4.cDNA ligation adapter
5.cDNA separation according to length
6.CDNA and yeast two-hybrid library vector (pGADT7 or pDEST22) for all-direct recombination
7. Electroconversion of recombinant products
8. Library detection and plasmid extraction
3.1 Comparison and advantages of All-direct method and SMART method
3.2? Comparison and advantages of All-direct method and Gateway method
Within 25 working days, if yeast needs to be transformed, it will be extended by 15 working days.
Note 1: You can choose to add a homogenization step to this yeast library. We use the DSN homogenization method to construct a high-quality homogenized yeast hybrid library, which can greatly reduce the workload of subsequent yeast screening. and screening efficiency, etc.
To construct a designated yeast two-hybrid cDNA library, the customer can provide us with tissues, cells or total RNA:
Cell sample: the number of cells is greater than 1*10^7
Animal samples: greater than 1g
Plant samples: greater than 2g
Total RNA: greater than 200ug
Currently we have successfully completed thousands of Library construction without samples, the main customers are Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai Jiao Tong University, Zhejiang University, Zhejiang Academy of Agricultural Sciences, Shandong Academy of Agricultural Sciences and many other units to complete yeast double (single) hybrid library construction services , species include humans, mice, rats, rice, Arabidopsis, peony, rice planthoppers, apples, rape, wheat, shells, grapes, fungi, etc.
We are one of the few companies in China that can provide both library construction and library screening. For yeast two-hybrid screening services, you only need to provide the bait gene sequence. We will perform the following work, and the screening quotation is 30,000 yuan:
1. Sequencing verification of the bait vector and its construction into the double-hybrid screening vector
2. Self-activation detection of the bait gene. After passing the test, proceed to the next step of the experiment .
3. Transform yeast cells with bait plasmid, and then transform into library plasmid after verification
4. Yeast screening and 3AT condition optimization
5. Sequencing of screening results
6. Reversal verification of screening results
7. Submission of screening results: including screening report, sequencing results and blast results of screening results, and physical plasmids cloned from screening results.
9.1. How to choose the appropriate library construction method for library construction?
※ We are the only company on the market that can provide 3 library construction methods at the same time, which is enough to show the technical strength of our company.
※ The comparison of the 3 methods is as follows:
? 1) Clonetech’s SMART method, this method is basically eliminated. We have a detailed technical comparison above, and its only The advantage is that the starting amount of RNA can be very small, generally only a few ug is needed. This method is very cost-effective and is generally not recommended.
? 2) Invitrogen's method is much better in quality than the SMART method. It also has very high quality requirements for reagents, and all original reagents from Invitrogen must be used. We all used Invitrogen's reagents for construction, and made some technical improvements to improve the quality of the library. This method has higher requirements for starting RNA, and the recommended starting dosage is greater than 200ug. However, this method has a serious shortcoming, that is, it requires two recombinations to obtain the yeast library. One more recombination will lead to one more library amplification process, which will seriously affect the quality of the library. There is a detailed technical comparison and introduction above.
? Other companies that use this method to build libraries can only use commercial kits to build libraries without making any improvements. And due to the purchase channel, they cannot buy some reagents in the kit, and the quality will be greatly reduced. For example, the transformation efficiency of DH10B electroporated competent cells is more than 100 times higher than that of domestically produced cells. This competent cell requires a lot of customs approval to purchase, and other companies cannot import it because they do not have relevant qualifications.
? 3) All-Direct patented method, this is our exclusive patented method, which is an important improvement on the invitrogen method and retains its advantages (no need for PCR amplification, high library fidelity) , eliminating the disadvantage of requiring two recombinations and greatly improving library quality. Compared with the invitrogen method, the average length can be increased by 200bp, and the library capacity can be increased by more than 10 times. This method requires the same amount of RNA as the invitrogen method.
9.2. What are the product contents of the yeast double hybrid library provided by Baoji Biotech?
※ The library products we provide include library quality (greater than 200ug) and E. coli library glycerol bacteria. At the same time, you can choose to provide a library of glycerol bacteria transformed into yeast cells. We will provide 100 library glycerol bacteria transformed into yeast cells, which can be used for 100 times of library screening.
※ Among them, the library plasmid can be screened using the ***transfer method, and the yeast cell library glycerol can be screened using the maiting method. You can choose freely according to the teacher's experimental habits, and there will be no difference in the results. of.
9.3. How to check library quality?
※ For library quality, a simple gold standard is that all genes in the sample are in the library, and more than half of the genes must be full-length genes. This library is qualified.
※ For library testing, the products we deliver can be tested in the following aspects:
Based on the teacher’s sample information, select 3-5 low-copy genes , design synthetic primers, use the library plasmid we provide as a template, and perform PCR amplification. If low-copy genes can be amplified, high-copy genes will not be lost, and the library quality can be judged to be qualified.
※ For the E. coli library glycerol bacteria we provide, you can perform gradient dilution and plate culture, calculate the library capacity the next day, and select single clones for PCR amplification and sequencing to determine the inserted fragment. Length and empty rate.
9.4. How to evaluate library quality?
※ The key indicators of library quality are mainly library capacity, blank rate and average length of inserted fragments.
※ The higher the original library capacity of the library, the better. Our company promises a library capacity greater than 1*10^7. This is the original library capacity, that is, without any amplification process, directly The converted library capacity. It is more than 10 times higher than the 1*10^6 library capacity promised by other companies. The following is a detailed explanation of the impact of library capacity on library quality:
※ Biological samples, except for lower organisms, are currently The number of genes in more studied species, such as humans, rats, and mice, is around 5*10^4, and the library must have at least 100-fold coverage, which means that the library capacity needs to be at least 5*10^6.
※ For plant samples, the number of genes is much larger than that of humans. For example, the number of genes in rice samples is 2-3 times that of humans, and the number of genes in wheat samples is 5-6 times that of humans. This way More library capacity is required. For rice samples, 100 times the number of genes needs to be covered, and the required original library capacity is greater than 1*10^7. Libraries provided by other companies on the market can only cover about 10 times at most. Such low coverage will inevitably lead to the loss of a large number of genes.
※ Some companies promote that the higher the library capacity, the better. This is definitely irresponsible deception and makes unreasonable excuses for the substandard quality of their libraries.
※ As for the length of the inserted gene, other companies generally have an average length of around 800bp. Our company can promise to achieve more than 1200bp. This gap is huge. Below, we will introduce in detail the impact of the length of the inserted fragment on the quality of the library. The impact of The 5'UTR region, 3'UTR region and polyA region, combined, the length of the gene is generally at least 1000bp. Since the library is a mixture, there will be competitive suppression. If there are too many short genes, it will inevitably affect the proportion of long genes and the expression abundance of long genes.
※ For our library products, the average insert length will be more than 1200bp, and the shortest one will be around 1000bp. For library screening, it does not mean that only one fragment of the gene is enough. The function of the protein requires the participation of multiple factors. If the inserted gene is too short, the screened result will only be a gene fragment, or even a small fragment of the gene close to polyA. How feasible is this result? Basically it can be considered a false positive result.
※ Some companies claim that the longer the gene insert in the library is not, the better. In fact, it is an extremely irresponsible and unreasonable deception to customers, just because they do not have the technology to make it longer. Clip length, while looking for reasons.
9.5. Which screening method is better, mating or *** transfer?
The results of the two methods of ***transfer and maiting are the same. They are just a method of transferring a library plasmid to yeast cells. There is no difference in the subsequent library screening. You can follow the teacher's experimental habits. Freely chosen.