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