Finding Full-Length Targets With Uncut cDNA Libraries

Uncut cDNA libraries deliver accelerated results. Using special recombination techniques, we can construct comprehensive, representative uncut cDNA libraries rapidly (Table 1). Because restriction digestion bias is eliminated, you don’t have to worry that precious target sequences will be truncated. Our uncut cDNA libraries have:

  • >90% recombinants
  • High percentage of full-length clones
  • Very high cloning efficiency

Table 1. Sample data for uncut cDNA libraries.

Fractionation method Total clones sequenced Insert range (kb) Average insert size (kb) Full-length ORF
column380.25-4.51.928 (74%)
gel at 3.5 kb and up360.65-7.12.812 (33%)

We routinely obtain 5–10 x 106 clones from 30 ng of fractionated cDNA. Maximize your chance of obtaining full-length target sequences by choosing our uncut library option.

Microquantity Libraries: Scarce Samples Are Not a Problem

The ability to make cDNA libraries from small amounts of RNA (as little as 100 µg) or tissue (as few as 100 mg) 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 RNA. The technology is not PCR-based.

Nanoquantity Libraries: Really Scarce Samples Are Not a Problem, Either

Even the smallest samples can be used to generate a cDNA library of surprising complexity and high average insert size. Invitrogen’s proprietary nanoquantity mRNA isolation technology significantly increases the quality and quantity of mRNA that is obtained from precious and scarce starting materials (Table 2). Combined with highly efficient recombination cloning technology, highly representative nanoquantity cDNA libraries can be constructed using as little as 10 µg of RNA or 10 mg of tissue, without PCR amplification.

Table 2. Construction of nanoquantity cDNA libraries from different amounts of tissue/cell/mRNA.

Amount of tissue/cell/mRNA Average insert size (kb) % insert Titer (cfu/ml) Total primary cfu
1 mg Tissue (Rat Brain)1.3100%0.11 x 1061.30 x 106
5 mg Tissue (Rat Brain)1.8100%1.30 x 10615.6 x 106
5 x 104 cells (293 Cell)1.391%0.18 x 1062.16 x 106
5 x 105 cells (293 Cell)1.6100%6.60 x 10679.0 x 106
5 ng mRNA (HeLa Cell)1.491%0.04 x 1060.48 x 106
10 ng mRNA (Invertebrate Embryo)2.2100%0.03 x 1060.36 x 106
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