SuperScript® Full-Length cDNA Library Construction Kit
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
|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 x105||48||~1.3||12||92%|
|Template-Switching (Competitor’s kit)||0.51 x 105||32||~0.6||11||55%|
|SuperScript® Full-Length||214 x 105||48||~1.4||14||100%|
The SuperScript® Full-Length cDNA Library Construction Kit proves to be a superior method to obtain full length clones.
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.