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    Item# Description List Price Web Price Qty
    M0368L ProtoScript II Reverse Transcriptase - 10,000 units $246.00
    $221.40
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    M0368S ProtoScript II Reverse Transcriptase - 4,000 units $123.00
    $110.70
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    M0368X ProtoScript II Reverse Transcriptase - 40,000 units $882.00
    $793.80
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    *On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca
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    Product Introduction

    • Recombinant M-MuLV reverse transcriptase
    • Reduced RNase H activity
    • Increased thermostability

    Product Information

    Description

    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, higher yield of cDNA and more full-length cDNA product up to 12 kb.

    Protoscript II Reverse Transcriptase performs as well as other RNase H Reverse Transcriptases Protoscript II Reverse Transcriptase performs as well as other RNase H– Reverse Transcriptases

    Jurkat total RNA (1 μg) was used in a 20 μl first strand cDNA synthesis. Mixtures of all reaction components, except for reverse transcriptase, were held at different temperatures for 3 min. 200 units SuperScript® II (A) or NEB’s ProtoScript II Reverse Transcriptase (B) was added and incubated at the indicated temperature for 50 minutes, followed by heat inactivation for 5 min at 80°C. 1 μl of cDNA was used in a 25 μl PCR using LongAmp Taq Master Mix (NEB #M0533) for 35–40 cycles. Ladder L is the 2 Log DNA Ladder (NEB #N0469). 
    Robust cDNA synthesis is achieved even with longer templates Robust cDNA synthesis is achieved even with longer templates

    Jurkat total RNA (1 μg) was used in a 20 μl first strand cDNA synthesis with 200 units of NEB ProtoScript II Reverse Transcriptase. Reactions were incubated at 42°C for 50 minutes, followed by heat inactivation for 5 minutes at 80°C. 1 μl of cDNA was used in a 25 μl PCR using LongAmp Taq Master Mix (NEB #M0533) for 35–40 cycles. Sizes are indicated above gel. 
    Generate high quality cDNA even with very low amounts of starting RNA Generate high quality cDNA even with very low amounts of starting RNA

    Decreasing amounts of Jurkat total RNA (1 μg – 1 pg) were used in 20 μl first strand cDNA synthesis with 200 units of NEB ProtoScript II Reverse Transcriptase. Reactions were incubated at 42°C for 50 minutes, followed by heat inactivation for 5 minutes at 80°C. 1 μl of cDNA was used in a 25 μl PCR using LongAmp Taq Master Mix (NEB #M0533) for 40 cycles. The target is a 0.6 kb fragment of GAPDH. Ladder L is the 2-Log DNA Ladder (NEB #N0469). 
    ProtoScript II Reverse Transcriptase displays superior sensitivity ProtoScript II Reverse Transcriptase displays superior sensitivity

    Decreasing amounts of luciferase mRNA (109 to 102) molecules were converted into cDNA in the presence of 1 ng Jurkat total RNA using 50 units of NEB ProtoScript II Reverse Transcriptase in a total reaction volume of 20 μl. 1/20 of the cDNA product was amplified using SsoAdvanced™ SYBR® Green Supermix. As few as 5 molecules of luciferase mRNA are detectable.

    Product Source

    The gene encoding a mutant M-MuLV Reverse Transcriptase (RNase H) is expressed in E. coli and purified to near homogeneity.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C) Concentration
    ProtoScript® II Reverse Transcriptase Reaction Buffer -20 5 X
    DTT (100mM) -20 10 X
    Product Categories:
    RT-PCR Products,
    Reverse Transcriptases & RT-PCR Products,
    cDNA Synthesis & Reverse Transcriptases Products
    Applications:
    cDNA Synthesis,
    Reverse Transcription (cDNA Synthesis),
    RT-PCR & cDNA Synthesis
    ,
    RT-qPCR, RT-PCR and cDNA Synthesis
    ,
    PCR

    Properties and Usage

      Unit Definition

      One unit is defined as the amount of enzyme that will incorporate 1 nmol of dTTP into acid-insoluble material in a total reaction volume of 50 μl in 10 minutes at 37°C using poly(rA)•oligo(dT)18 as template.

      Reaction Conditions

      1X ProtoScript® II Reverse Transcriptase Reaction Buffer
      Incubate at 42°C

      1X ProtoScript® II Reverse Transcriptase Reaction Buffer
      50 mM Tris-HCl
      75 mM KCl
      3 mM MgCl2
      (pH 8.3 @ 25°C)

      Storage Temperature

      -20°C

      Storage Conditions

      20 mM Tris-HCl
      100 mM NaCl
      1 mM DTT
      0.1 mM EDTA
      50% Glycerol
      0.01% IGEPAL® CA-630
      pH 7.5 @ 25°C

      Heat Inactivation

      65°C for 20 min

      Unit Assay Conditions

      50 mM Tris-HCl (pH 8.3), 75 mM KCl, 6 mM MgCl2, 10 mM dithiothreitol, 0.01% IGEPAL CA-630, 0.5 mM dTTP, 0.4 mM poly(rA)•oligo(dT)18.

      Notes

      1. Reaction Conditions:
        1X ProtoScript II Reverse Transcriptase Reaction Buffer, 10 mM DTT, 200 units ProtoScript II Reverse Transcriptase, supplemented with 0.5 mM dNTPs (not included) and 5 µM dT23VN (not included). Incubate at 42°C for 50 minutes. If random primers are used, a 10 minute incubation at room temperature is recommended before transferring to 42°C.

      References

      1. Roth, M.J., Tanese, N. and Goff, S.P. (1985). J. Biol. Chem.. 260, 9326-9335.
      2. Kotewicz, M.L. et al, (1988). Nuc. Acids Res.. 16, 265-277.
      3. Lim, D. et al, (2006). J. Virol.. 80, 8379-8389.
      4. Sambrook, J., Fritsch, E.F. and Maniatis, T. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.(Ed.), Molecular Cloning: A Laboratory Manual. 1989, pp. 5.52-5.55, 8.11-8.17.

      Faqs & Tech Tips

      FAQs

      1. What is the difference between NEB# M0368 and NEB# M0253?
      2. What is the optimal reaction temperature for ProtoScript II Reverse Transcriptase (M0368)?
      3. Can the cDNA products be used in real-time PCR analysis?
      4. What thermostable DNA polymerase can be used for PCR after cDNA synthesis?
      5. How can the yield be improved when using ProtoScript II Reverse Transcriptase?
      6. How can the length of the product generated by M-MuLV Reverse Transcriptase be increased?
      7. Is RNaseH treatment required before PCR amplification?

      Protocols & Manuals

      Protocols

      1. First Strand cDNA Synthesis (Quick Protocol) (NEB #M0368)
      2. First Strand cDNA Synthesis (Standard Protocol) (NEB #M0368)
      3. First Strand cDNA Synthesis (No-RT Negative Control Reaction) (NEB #M0368)

      Datacards

      The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at [email protected] or fill out the Technical Support Form for appropriate document.)

      Other Tools & Resources

      Selection Charts

      • cDNA Synthesis Selection Chart
      • DNA Polymerase Selection Chart

      Usage & Guidelines

      • cDNA/Reverse Transcriptase Tips

      Citations

      • Terry Fei Fan Ng, Nikola O Kondov, Xutao Deng, Alison Van Eenennaam, Holly L Neibergs, Eric Delwart (2015) A metagenomics and case-control study to identify viruses associated with bovine respiratory disease. J Virol; 89, 5340-9. PubMedID: 25740998 , DOI: 10.1128/JVI.00064-15

      Quality & Safety

      Quality Assurance Statement

      Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

      Specifications

      The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

      Certificate Of Analysis

      The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

      Safety DataSheets

      The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

      ProtoScript® II Reverse Transcriptase
      ProtoScript® II Reverse Transcriptase Reaction Buffer
      DTT (100mM)