Full-Length Inserts

Finding full-length target sequences is a key objective of many library screens. Our novel full-length cDNA synthesis procedure combines a proprietary process for reducing RNase H reverse transcriptase activity with unique techniques for mRNA isolation, 5´ cap full-length enrichment, and reduction of oligo(dT) priming. Together, these steps greatly enhance the likelihood of constructing a library with full-length inserts (Figure 1 and Table 1), giving you better targets at which to aim.

Uncut Libraries and the Power of Gateway® Technology

To further enhance the chances of identifying full-length inserts, we have combined our proprietary full-length library procedure with the Gateway® recombination-based cloning technology. Using Gateway® technology allows us to avoid restriction enzyme digestion of the cDNA during library construction. Stringent quality control ensures that libraries are highly enriched for full-length cDNA clones (Figure 1 and Table 1). Our full-length cDNA libraries routinely achieve:

  • 65–85% full-length clones (Table 2)
  • >1 x 107 clones
  • Insert lengths up to 8 kb
Percent Full Length Clones
Figure 1. Estimation of the percentage of full-length inserts in six different cDNA libraries produced using Invitrogen’s proprietary full-length cDNA library technologies.

Table 1. Sample data for full-length human brain library. N=20

Gene Size(kb) Full-length clones
EIF4E2.0100
Protein tyrosine kinase3.071
TOP 33.780
Helix loop helix4.080
20XDH4.181
MAP45.572

Table 2. Comparison of uncut full-length (FL) and full-length (FL) cDNA libraries.

Major Difference Uncut FL Library FL Library
VectorPENTR222PCMVSport6.1 or pcDNA3.1
Starting Material (mRNA)2-6ug15ug
Res. Enzyme (Not I) CutNoYes
Cloning StrategyBxP RecombinationLigation
NormalizableNoYes

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