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standard cDNA libraries are made using our patented SuperScript® III reverse transcriptase enzyme.
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cDNA Library Construction
Our standard cDNA libraries are made using our patented SuperScript® III reverse transcriptase enzyme. Currently, first strand synthesis is primed with an oligo dT adapter or random primer and second strand synthesis is a modification of the Gubler and Hoffman method 1. Directionally cloned cDNA inserts facilitate the construction of subtracted or normalized cDNA libraries into pCMV·Sport 6.1 or pcDNA™3.1 vectors. After transformation into high efficiency competent E. coli, we guarantee at least 3 x 106 primary clones with an average insert size of at least 1kb. Unlike libraries constructed using random cloning procedures, all of the clones in a directional library are properly oriented for expression, antibody screening, or isolation of specific cDNA clones using the GeneTrapper® cDNA Positive Selection System.
Our standard cDNA libraries are made using our patented SuperScript® III reverse transcriptase enzyme. Currently, first strand synthesis is primed with an oligo dT adapter or random primer and second strand synthesis is a modification of the Gubler and Hoffman method 1. Directionally cloned cDNA inserts facilitate the construction of subtracted or normalized cDNA libraries into pCMV·Sport 6.1 or pcDNA™3.1 vectors. After transformation into high efficiency competent E. coli, we guarantee at least 3 x 106 primary clones with an average insert size of at least 1kb. Unlike libraries constructed using random cloning procedures, all of the clones in a directional library are properly oriented for expression, antibody screening, or isolation of specific cDNA clones using the GeneTrapper® cDNA Positive Selection System.
Vector List:
pCMV·Sport 6.1
pcDNA™3.1
pcDNA™3.1
Libraries can be constructed in your vector as well. Depending on the characteristics of your vector, some modification of it may be necessary. If you require other options, Contact an Invitrogen Custom Services Representative.
1. Gubler U, Hoffman BJ (1983), Gene 25(2-3):263-9
1. Gubler U, Hoffman BJ (1983), Gene 25(2-3):263-9
Libraries constructed from microquantities of RNA
The ability to make cDNA libraries from small amounts of mRNA (as little as 200 nanograms) or cells (as little as 1 million) is the hallmark of our latest generation of cDNA technologies, microquantity cDNA synthesis. This technology, in conjunction with our new nanoquantity mRNA isolation system and patented SuperScript® reverse transcriptase allows for the construction of directionally cloned cDNA libraries from rare and small tissue samples, cultured cells or mRNA. The technology is not PCR based.
The ability to make cDNA libraries from small amounts of mRNA (as little as 200 nanograms) or cells (as little as 1 million) is the hallmark of our latest generation of cDNA technologies, microquantity cDNA synthesis. This technology, in conjunction with our new nanoquantity mRNA isolation system and patented SuperScript® reverse transcriptase allows for the construction of directionally cloned cDNA libraries from rare and small tissue samples, cultured cells or mRNA. The technology is not PCR based.
Library Normalization and Subtraction
The enrichment of cDNA libraries for rare and unique sequences is accomplished by using our library normalization procedure (patent pending), which reduces the frequency of abundant sequences as much as 100 to 200 times and increases the frequency of rare sequences in a cDNA library. Independent sequencing of these normalized libraries indicates that greater than 30% of the clones in these libraries are unique when compared to the public database of more than two million ESTs and fifty percent of the clones have been seen no more than five times. The normalization process results in little reduction of the average insert size of cDNA, and the libraries have greater than 95% recombinant clones.
The enrichment of cDNA libraries for rare and unique sequences is accomplished by using our library normalization procedure (patent pending), which reduces the frequency of abundant sequences as much as 100 to 200 times and increases the frequency of rare sequences in a cDNA library. Independent sequencing of these normalized libraries indicates that greater than 30% of the clones in these libraries are unique when compared to the public database of more than two million ESTs and fifty percent of the clones have been seen no more than five times. The normalization process results in little reduction of the average insert size of cDNA, and the libraries have greater than 95% recombinant clones.
Using customer-supplied tissue or total RNA, a primary library is prepared and amplified, followed by normalization. The resulting library is amplified and quality controlled. Additional steps are taken throughout the process to ensure a high-quality library. You can access this technology by ordering a custom normalized library, licensing our technology and bringing it into your laboratory or by ordering from a growing list of normalized libraries that we have already produced. Get more information at the NCI CGAP web site on the quality of some of these libraries.
We also offer a custom cDNA library subtraction service. If you are interested in removing the vascular component of a particular tissue or comparing the gene expression in induced and uninduced cell lines, then this technology may meet your needs. Contact us and we will be happy to share our most recent data on this technology.
Library Amplification
We use a semi-solid agarose protocol for amplification, ensuring that each colony has an equal chance of growing, thereby faithfully preserving the representation of the primary library and increasing the number of colony forming units by at least 1000 fold.
We use a semi-solid agarose protocol for amplification, ensuring that each colony has an equal chance of growing, thereby faithfully preserving the representation of the primary library and increasing the number of colony forming units by at least 1000 fold.
Selecting Competent Cells for Transformation
To produce a large number (3 x 106) of independent clones, the plasmid-cDNA ligations are electroporated into Electromax® DH10B™ Competent Cells. The DH10B™ bacterial strain is the preferred choice for high-throughput sequencing of cDNA libraries. Unless you have a specific request, we will use Electromax DH10B® Competent Cells.
To produce a large number (3 x 106) of independent clones, the plasmid-cDNA ligations are electroporated into Electromax® DH10B™ Competent Cells. The DH10B™ bacterial strain is the preferred choice for high-throughput sequencing of cDNA libraries. Unless you have a specific request, we will use Electromax DH10B® Competent Cells.
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