SuperScript® Full-Length cDNA Library Construction Kit
The SuperScript® Full-Length cDNA Library Construction Kit is the superior choice for generation of full-length cDNA libraries. The kit utilizes a simple and robust protocol for enrichment of 5’-CAPed mRNA which prevents truncated mRNA from being reverse-transcribed, followed by transfer of intact double-stranded cDNAs directly into the library vector using Gateway® recombination cloning.

The SuperScript® Full-Length cDNA Library Construction Kit ensures:

  • A simple, easy to follow protocol without the complicated steps of do-it-yourself ‘CAP trapper’ cDNA library construction methods
  • Creation of a cDNA library with transcripts selected for full-length- from total RNA or enriched mRNA (98–100% full length clones)
  • Highly efficient cloning of the double stranded cDNA sequences into a Gateway® recombination cloning vector avoids low-efficiency restriction digestions and ligation steps.
  • Construction of cDNA libraries ready for single step transfer into multiple Gateway® expression vectors.

Why do you need a Full-Length cDNA Library Construction Kit?

  • Preparation of next-generation sequencing templates from transcripts selected for full-length
  • Identification of the exact location of transcription start sites
  • Discovery of different gene isoforms, or alternatively spliced genes, that are expressed in different cells/tissues
  • Discovery of sequence motifs that reside near the 5’ end of the gene
  • Isolation of full DNA sequences supporting identification of gene families, and thus discovery of new family members without further labor-intensive procedures such as 5’ RACE

 

 

 

 

Proven results

The table below shows data comparing the results for three methods for full-length library preparation using mRNA samples spiked with 25% of rabbit hemoglobin capped mRNA. At least 48 clones from each library were sequenced following the library construction.

Method Total primary cfu Total clones w/good seq. Average insert size (Kbp) Clones for rabbit hemoglobin % Full-length of RHG*
Cap-Trapper (home brewed)118 x10548~1.31292%
Template-Switching (Competitor’s kit)0.51 x 10532~0.61155%
SuperScript® Full-Length214 x 10548~1.414100%

The SuperScript® Full-Length cDNA Library Construction Kit proves to be a superior method to obtain full length clones.

How does the kit work?

1st strand cDNA is synthesized at high temperature (50-55ºC) using SuperScript® Reverse Transcriptase III and an anchored oligo-dTnVN primer containing a Gateway® attB2 recombination sequence. The mRNA-cDNA hybrids are treated with RNAse I, which exclusively digests single strand RNA eliminating the cap structure (m7GpppG) from any incompletely synthesized (non full-length) cDNA-mRNA hybrids. Full-length mRNA-cDNA hybrids are captured using a cap-antibody conjugated to magnetic beads. Unbound non-full-length mRNA-cDNA hybrids are washed away. Enriched 1st strand full-length cDNAs are eluted by NaOH and ligated to a Gateway® attB1 adaptor followed by primer extension using high-fidelity DNA polymerase. Gateway® adapted double strand cDNAs are cleaned and sized through a column to exclusively remove the adaptor and primers. Clean double strand cDNAs are cloned with high efficiency into a Gateway® pDONR 222 vector using Gateway® BP Clonase and transformed into bacteria by electroporation.