ProtoScript II First Strand cDNA Synthesis Kit features two optimized mixes,ProtoScript II Enzyme Mix and ProtoScript II Reaction Mix. The enzyme mixcombines ProtoScript II Reverse Transcriptase and Murine RNase Inhibitor,while the reaction mix contains dNTPs and an optimized buffer. ProtoScriptII Reverse Transcriptase is a recombinant M-MuLV reverse transcriptasewith reduced RNase H activity and increased thermostability. It can be usedto synthesize first strand cDNA at higher temperatures than the wild typeM-MuLV. The enzyme is active up to 48°C, providing higher specificity andhigher yield of cDNA.The kit also provides two optimized primers for reverse transcription andnuclease-free water. An anchored Oligo-dT primer [d(T)23VN] forces the primerto anneal to the beginning of the polyA tail. The optimized Random PrimerMix provides random and consistent priming sites covering the entire RNAtemplates including both mRNAs and non-polyadenylated RNAs. The firststrand cDNA product generated is up to 10 kb.Figure 1: cDNA Synthesis of Jurkat RNA with the ProtoScript II FirstStrand cDNA Synthesis KitFirst strand cDNA synthesis was carried out in the presence of 1X ProtoScript II Reaction Mix and 1XProtoScript II Enzyme Mix at 42°C using 250 ng of Jurkat total RNA. Four amplicons from differentgenes were amplified using 1X LongAmp® Taq 2X Master Mix (NEB #M0287). Lane 1, a 1.9 kbamplicon from SDHA gene; Lane 2, a 5.5 kb from the guanine nucleotide exchange factor; Lane 3,a 7.3 kb amplicon from Xrn-1 gene; and Lane 4, a 9.2 kb amplicon from fibrillin gene. Marker M isthe 1 kb Plus DNA Ladder (NEB #N3200).Quantitative detection of reverse transcription products using the ProtoScript II First Strand cDNA Synthesis KitTen-fold serial dilutions of in vitro transcribed luciferase RNA (1x102 to 1x109 copies) were mixed with 1 ng Jurkat total RNA. The RNA was reverse transcribed using first strand cDNA synthesis kits, as indicated, and 1/10th of the cDNA product was used for qPCR using luciferase-specific primers and iTaqTM Universal SYBR Green Supermix (BioRad). Ct values were plotted as a function of input luciferase RNA equivalent copies. Each data point represents the mean +/- 95% confidence interval of a minimum of 5 amplification reactions. ProtoScript II – NEB ProtoScript II First Strand cDNA Synthesis Kit and random primer mix; SuperScript II – Invitrogen SuperScript First Strand Synthesis System for RT-PCR and random hexamers; SuperScript® III – Invitrogen SuperScript III First Strand Synthesis SuperMix and random hexamers.
This product is related to the following categories:
RT-PCR Products,
Reverse Transcriptases & RT-PCR Products,
cDNA Synthesis & Reverse Transcriptases Products
This product can be used in the following applications:
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