ProtoScript® II Reverse Transcriptase |NEB酶试剂 New England Biolabs

发表于由

上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

产品信息

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 |
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 ProtoScript® II Reverse Transcriptase |

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 ProtoScript® II Reverse Transcriptase |

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 |
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.

产品来源

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

产品类别:
RT-PCR Products,
cDNA Synthesis & Reverse Transcriptases Products

应用:
Helicase-dependent Amplification,
cDNA Synthesis,
Reverse Transcription (cDNA Synthesis),

RT-PCR & cDNA Synthesis,

PCR

  • 产品组分信息

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • M0368S     -20    
        ProtoScript® II Reverse Transcriptase M0368SVIAL -20 1 x 0.02 ml 200,000 units/ml
        ProtoScript® II Reverse Transcriptase Reaction Buffer B0368SVIAL -20 1 x 1.5 ml 5 X
        DTT B1034AVIAL -20 1 x 0.5 ml 0.1 M
    • M0368L     -20    
        ProtoScript® II Reverse Transcriptase M0368LVIAL -20 1 x 0.05 ml 200,000 units/ml
        ProtoScript® II Reverse Transcriptase Reaction Buffer B0368SVIAL -20 1 x 1.5 ml 5 X
        DTT B1034AVIAL -20 1 x 0.5 ml 0.1 M
    • M0368X     -20    
        ProtoScript® II Reverse Transcriptase M0368LVIAL -20 4 x 0.05 ml 200,000 units/ml
        ProtoScript® II Reverse Transcriptase Reaction Buffer B0368SVIAL -20 4 x 1.5 ml 5 X
        DTT B1034AVIAL -20 4 x 0.5 ml 0.1 M

  • 特性和用法

    单位定义

    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.

    反应条件

    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)

    贮存溶液

    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

    热失活

    65°C for 20 minutes

    单位活性检测条件

    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.

  • 相关产品

    相关产品

    • e6560-protoscript-ii-first-strand-cdna-synthesis-kit
    • Oligo d(T)23 VN
    • Oligo d(T)18 mRNA 引物
    • 小鼠 RNase 抑制剂
    • dNTP 混合液
    • dNTP 套装
    • 南极热敏 UDG
    • NEBNext® Ultra II RNA 非链特异性第二链合成模块

  • 注意事项

    1. 反应条件：
      1X ProtoScript II 反转录酶反应缓冲液、10 mM DTT、200 单位 ProtoScript II 反转录酶，添加 0.5 mM dNTP（不随试剂盒提供）和 5 µM dT23VN（不随试剂盒提供）。42℃ 温育 50 分钟。如果使用随机引物，推荐先在室温下温育 10 分钟，再进行 42℃ 反应。

  • 参考文献

    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.

操作说明、说明书 & 用法

  • 操作说明

    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)

  • 使用指南

    • cDNA/Reverse Transcriptase Tips

工具 & 资源

  • 选择指南

    • DNA Polymerase Selection Chart

FAQs & 问题解决指南

  • 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?
    8. What is the difference between Induro Reverse Transcriptase (NEB #M0681) and ProtoScript II Reverse Transcriptase (NEB #M0368)?
    9. Why do I have low cDNA yields?
    10. How do I know whether my template RNA is of good quality?