ProtoScript® II First Strand cDNA Synthesis Kit

The ProtoScript® II First Strand cDNA Synthesis Kit is optimized mix for higher specificity and yield of cDNA.

  • Recombinant M-MuLV reverse transcriptase with reduced RNase H activity and increased thermostability (active up to 48°C)
  • Generates cDNA up to 10 kb
  • Supplied with Oligo-dT primers, Randomized Primer Mix, and nuclease-free water

Ordering Information

E6560S_pdp_800X450
  • 30 reactions
    Cannot order from your location. Contact NEB for order information
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  • 150 reactions
    Cannot order from your location. Contact NEB for order information
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  • Product Information
    ProtoScript II First Strand cDNA Synthesis Kit features two optimized mixes, ProtoScript II Enzyme Mix and ProtoScript II Reaction Mix. The enzyme mix combines ProtoScript II Reverse Transcriptase and Murine RNase Inhibitor, while the reaction mix contains dNTPs and an optimized buffer. ProtoScript II Reverse Transcriptase is a recombinant M-MuLV reverse transcriptase with reduced RNase H activity and increased thermostability. It can be used to synthesize first strand cDNA at higher temperatures than the wild type M-MuLV. The enzyme is active up to 48°C, providing higher specificity and higher yield of cDNA.

    The kit also provides two optimized primers for reverse transcription and nuclease-free water. An anchored Oligo-dT primer [d(T)23VN] forces the primer to anneal to the beginning of the polyA tail. The optimized Random Primer Mix provides random and consistent priming sites covering the entire RNA templates including both mRNAs and non-polyadenylated RNAs. The first strand cDNA product generated is up to 10 kb.

    Figure 1: cDNA Synthesis of Jurkat RNA with the ProtoScript II First Strand cDNA Synthesis KitFigure 1: cDNA Synthesis of Jurkat RNA with the ProtoScript II First Strand cDNA Synthesis Kit

    First strand cDNA systhesis was carried out in the presence of 1X ProtoScript II Reaction Mix and 1X
    ProtoScript II Enzyme Mix at 42°C using 250 ng of Jurkat total RNA. Four amplicons from different
    genes were amplified using 1X LongAmp® Taq 2X Master Mix (NEB #M0287). Lane 1, a 1.9 kb
    amplicon 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 is
    2-Log DNA Ladder (NEB #N3200).
    Quantitative detection of reverse transcription products using the ProtoScript II First Strand cDNA Synthesis KitQuantitative detection of reverse transcription products using the ProtoScript II First Strand cDNA

    Ten-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.

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C) Concentration
    Nuclease-free Water -20
    ProtoScript® II Reaction Mix -20 2 X
    ProtoScript® II Enzyme Mix -20 10 X
    Oligo d(T)23 VN -20 50 μM
    Random Primer Mix -20 60 μM
    Product Categories:
    RT-PCR Products,
    Reverse Transcriptases & RT-PCR Products,
    cDNA Synthesis & Reverse Transcriptases
    Applications:
    cDNA Synthesis,
    Reverse Transcription (cDNA Synthesis),
    RT-PCR & cDNA Synthesis
    ,
    RT-qPCR, RT-PCR and cDNA Synthesis
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