Fast, Economical, and Straightforward

Although standard by name, this service employs many of the superior products and process improvements that make our cDNA libraries the highest quality. Choose this service when you need fast turnaround and have straightforward screening and downstream needs.

The Standard Service

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 a random primer, and second-strand synthesis is a modification of the Gubler and Hoffman method.* 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 1 kb. 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.

Standard libraries can also be constructed in a custom vector of your choosing. In these cases, and depending on the characteristics of the vector, some modifications of the vector may be necessary, with impacts on the project timeline.

In addition to this standard process, we offer a number of options to maximize the quality and usefulness of your library:

  • Transcript Enrichment Through Normalization and Subtraction
Enrichment for rare and unique sequences is accomplished using our proprietary normalization procedure (patent pending), which reduces the frequency of abundant sequences (as much as 100- to 200-fold) and increases the frequency of rare sequences. Independent sequencing of normalized libraries indicates that more than 30% of the clones in these libraries are unique when compared to the public Expressed Sequence Tags (EST) database. In addition, 50% of clones are observed no more than five times. The normalization process results in little reduction of the average insert size of the cDNA, and the libraries have more than 95% recombinant clones.

Using 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, by 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 on the quality of some of these libraries at the NCI CGAP website.

We also offer a custom cDNA library subtraction service. If you are interested in removing the vascular component of a particular tissue, or in comparing gene expression in induced and uninduced cell lines, then this technology may meet your needs.

  • Faithful Library Amplification
We use a semisolid agarose protocol for library amplification. This ensures that each colony has an equal chance of growing, which in turn helps to preserve the representation of the primary library. Amplification generally increases the number of colony forming units (CFUs) at least 1,000-fold.

  • Best Competent Cells for the Highest-Quality Library
To produce a guaranteed minimum number of independent clones (3 x 106), ligated cDNA is electroporated into Electromax® DH10B™ Competent Cells. The DH10B™ bacterial strain is the preferred choice for high-throughput sequencing of cDNA libraries. For maximum flexibility, however, we can use another strain should you so request.

*Gubler U, Hoffman BJ (1983) Gene 25(2-3):263–269.

For more information or to order a service, call 800.955.6288 x2 or e-mail custom.services@lifetech.com.