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Protocols for ProtoScript® First Strand cDNA Synthesis Kit FAQs for Polymerases Technical Reference for Polymerases Enzyme Finder NEBcutter NEBuffer Chart Double Digest Finder Isoschizomers DNA Sequences and Maps REBASE Deoxynucleotide Solution Mix M-MuLV Reverse Transcriptase M-MuLV Reverse Transcriptase Reaction Buffer Oligo d(T)23 VN Random Primer 9 RNase H RNase Inhibitor 1 kb DNA Ladder 100 bp DNA Ladder 6-Tube Magnetic Separation Rack Biotinylated Oligo d(T)18 mRNA Primer dsRNA Ladder polyA Spin mRNA Isolation Kit ssRNA Ladder Streptavidin Magnetic Beads

Home > Products > Polymerases > RT-PCR and qRT-PCR > ProtoScript® First Strand cDNA Synthesis Kit ProtoScript® First Strand cDNA Synthesis Kit Catalog # Size Concentration Price Qty   E6500L 150 reactions   $632.00 E6500S 30 reactions   $158.00 Prices are in US dollars and valid only for US orders. Download: Technical Bulletin|Manual|MSDS PDF Flexibility — choose appropriate PCR format Sensitivity — efficient reverse transcription from different starting RNA amounts Description: The ProtoScript® First Strand cDNA Synthesis Kit has been designed for the highly efficient production of cDNA copies of mRNA using Moloney-Murine Leukemia Virus (M-MuLV) Reverse Transcriptase (RT). The single-stranded cDNA can be used directly for PCR* amplification with gene-specific primers or for other downstream applications (Figure 1). ProtoScript is optimized for RT reactions using a wide range of total RNA amounts (10 pg-2 µg) (Figure 2). Both long (> 13 kb) and low abundance cDNAs can be detected by amplification after cDNA synthesis. The kit provides all the reagents required for performing first-strand cDNA synthesis reactions from total or polyA+-selected RNA. Two cDNA synthesis primers are provided, oligo-dT (dT23VN) which prevents priming at internal sites in polyA tails and random nonamers (dN)9. RNase H is included in the kit to remove RNA template strands after cDNA synthesis. This additional step improves amplification yields when low starting amounts of RNA are used for cDNA synthesis. Control total RNA as well as a set of positive control amplification primers are included. Note: V= A, G or C and N= A, G, C or T. Method Overview: Three alternatives in priming RNA for cDNA synthesis are: i) "oligo-dT" priming, ii) random priming, and iii) gene-specific priming (see Figure 1). Oligo-dT priming is used to take advantage of the poly-A tail found at the end 3´ end of most processed eukaryotic mRNAs. Priming cDNA synthesis with oligo-dT ensures that all cDNA copies terminate at the 3´ end of the mRNA and produces the longest contiguous cDNAs. Although the 5´ ends of especially long mRNAs are under-represented, this priming method is preferred for most applications and gives consistent results. ProtoScript includes a variation of the standard oligo dT primer (dT23VN) with a non-T priming 3´ end which forces the primer to anneal to the start of the poly-A tail, thereby preventing priming at internal sites in poly-A tails (1,2). The use of random primers (N9) provides priming throughout the length of RNA for unbiased representation of all regions. It does not, however, discriminate between mRNA (with poly-A 3´ ends) or other, non-polyadenylated RNAs (such as ribosomal and tRNA) and yields shorter cDNAs on average. Random priming is more sensitive to the ratio of primer to RNA concentrations, especially when low amounts of RNA template are used (3). For some applications, first-strand synthesis is performed with a primer specific for a particular target mRNA. The cDNA produced can be used only for amplification of that transcript; separate RT reactions are therefore required for each mRNA. This priming method gives good results when the amount of RNA is limiting (below 10 ng) and only one particular cDNA is desired. For most applications oligo-dT priming is recommended. From a high quality RNA template (such as the included control RNA) targets longer than 13 kb away from poly-A tail have been successfully detected by PCR following oligo-dT-primed cDNA synthesis (Detection of the GTP exchange factor p619 cDNA in rat liver total RNA amplified in 30 cycles following the ProtoScript First Strand Synthesis Protocol). Figure 1: Overview of First-Strand cDNA Synthesis using different priming methods. Kit Components: Control (GAPDH) Primer Set Control Total RNA (rat liver) Deoxynucleotide Solution Mix M-MuLV Reverse Transcriptase M-MuLV Reverse Transcriptase Reaction Buffer Nuclease Free dH2O Oligo d(T)23 VN Random Primer 9 RNase H RNase Inhibitor Storage Conditions Storage Temperature: -20°C Figure 2: PCR-amplified GAPDH cDNA starting from different amounts of total RNA. Each lane contains an aliquot of PCR product (35 cycles) synthesized from amounts of total RNA ranging from 2 µg to 10 pg. The last lane is from a control reaction without reverse transcriptase. Marker is 250 ng of 100 bp DNA Ladder (NEB #N3231). References Khan, S.A. et al. (1991) Nucl. Acids Res., 19, 1715. Liao, J. and Gong, Z. (1997) Biotechniques, 23, 368-370. Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual (3rd Ed.), 8,46-8.53 and 11.37-11.42. Reagents Sold Separately Deoxynucleotide Solution Mix M-MuLV Reverse Transcriptase M-MuLV Reverse Transcriptase Reaction Buffer Oligo d(T)23 VN Random Primer 9 RNase H RNase Inhibitor Companion Products 1 kb DNA Ladder 100 bp DNA Ladder 6-Tube Magnetic Separation Rack Biotinylated Oligo d(T)18 mRNA Primer dsRNA Ladder polyA Spin mRNA Isolation Kit ssRNA Ladder Streptavidin Magnetic Beads