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  • ProtoScript® II Reverse Transcriptase

    This product was formerly called M-MuLV Reverse Transcriptase (RNase H)
    recombinant unique buffer heat inactivation
    Catalog #SizeConcentrationPriceQtyAdd to Cart
    M0368S4,000 units200,000 units/ml$82.00Add to Cart
    M0368L10,000 units200,000 units/ml$164.00Add to Cart
    M0368X40,000 units200,000 units/ml$588.00Add to Cart
      
    Categories:
    cDNA Synthesis & Reverse Transcriptases,
    Reverse Transcriptases & RT-PCR Products,
    RT-PCR Products
    Applications:
    cDNA Synthesis,
    cDNA Synthesis & RT-PCR,
    Reverse Transcription (cDNA Synthesis)

    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.

    M0368
    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). 
    M0368
    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. 
    M0368
    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). 
    M0368
    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-205X
    DTT (100mM)10X

    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.

    Quality Control

    Quality Assurance Statement

    • ProtoScript II Reverse Transcriptase is tested for its ability to synthesize a 9.2 kb cDNA product from total RNA by RT-PCR approach.

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • Protein Purity (SDS-PAGE):
      The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.
    • RNase Activity (1 Hour Digestion):
      The product is tested in a reaction containing a RNA substrate.  After incubation for 1 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

    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.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    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 info@neb.com or fill out the Technical Support Form for appropriate document.
    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?
    1. First Strand cDNA Synthesis Kit using ProtoScript II Reverse Transcriptase (M0368)

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