The invitrogen superscript vilo cdna synthesis kit provides the high temperature capability of superscript iii reverse transcriptase in an optimized format for generating firststrand cdna for use in realtime quantitative rtpcr qrtpcr. System for cdna synthesis, invitrogen life technologies, pn 18090019. The high dntp concentration during first strand cdna synthesis is critical to the priming specificity of the protocol. Thaw all necessary components and place them on ice. Guide to using the smarter cdna synthesis protocol for pcrselect cdna. Dna hybrid in a random fashion, producing nicks in addition to those produced by the reverse transcriptase. Components of thermo scientific revertaid first strand cdna synthesis kit.
Extralong firststrand cdna synthesis future science. Superscript iv first strand synthesis kit invitrogenis. The smarter pcr cdna synthesis kit is an improved version of. Dnafree total rna from jurkat t cellleukemia line and mrc5. Reverse transcription of rna into cdna protocol osski. Mix rna sample and primer dt 23 vn in a sterile rnase free.
Superscript ii reverse transcriptase thermo fisher scientific. Purified rna must be free of salts, metal ions, ethanol and phenol to avoid inhibiting the cdna synthesis reaction. Superscript iii firststrand synthesis system for rtpcr. Alternative protocol for onecycle cdna synthesis from total rna. Digital transcriptome profiling using selective hexamer. Increase the temperature of firststrand reaction up to 55c. Add the following reagents into a sterile, nuclease free tube on ice in the. Protocol for cdna synthesis and qrtpcr cdna synthesis. Add reaction mix to annealed rna rt reaction setup use the measurements below to prepare your rt reaction, or enter your own parameters in the column provided. The 260280 and 260230 values of my rna samples are above 2. Reaction protocol incubate the complete reaction mix in a thermal cycler using. Superscript vilo cdna synthesis kit product information.
This protocol described synthesis of cdna from polya mrna. C recommendation for optimal results using storage and stability the iscript cdna synthesis kit the maximum amount of the cdna reaction that is. Firststrand cdna synthesis using superscript ii rt. I am using superscript iv first strand synthesis kit for making cdna from rna samples. Smarter pcr cdna synthesis kit user manual takara bio. This formulation provides enhanced cdna synthesis efficiency and can be. The cdna synthesis system can be used to produce doublestranded cdna by two procedures.
Superscript iii firststrand synthesis system for rtpcr author. Protocols cdna synthesis from total rna or mrna since the conditions described for the first strand reaction are optimized, do not change volumes and concentrations. Prior to cdna synthesis, please make sure that your rna is intact and free of. The protocol described in this article provides a classic and simple means to generate 3. Maintain an elevated temperature after the annealing step, as described in the protocol for cdna synthesis from highgc content transcripts, page 3.