cDNA文库构建
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.
pcDNA™3.1
1. Gubler U, Hoffman BJ (1983), Gene 25(2-3):263-9
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 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.
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.
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.
Invitrogen公司是在生命科学领域内最主要的供货商，其产品覆盖了自分子生物学研究、蛋白质表达、细胞培养、生物信息学、荧光检测及药品开发与筛选等生物工程的全过程。
自1987年成立以来，Invitrogen公司就一直致力于为生物科研及生产领域的客户提供最有效的工具，包括各种便于使用的试剂盒与消耗品，我们的目标是为客户提供更快捷、更可靠、更有效的产品及服务。
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