标签归档：transcriptase

WarmStart® RTx Reverse Transcriptase (Glycerol-free) |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

WarmStart RTx Reverse Transcriptase (Glycerol-free) is formulated without glycerol to support lyophilization, incorporation into microfluidic devices, and enable quick adoption into automation workflows.

WarmStart RTx Reverse Transcriptase is a unique in silico-designed RNA-directed DNA polymerase coupled with a reversibly-bound aptamer that inhibits RTx activity below 40°C. This enzyme can synthesize a complementary DNA strand initiating from a primer using RNA (cDNA synthesis) or single-stranded DNA as a template. RTx is a robust enzyme for RNA detection in amplification reactions and is particularly well suited for use in LAMP (Loop-mediated Isothermal Amplification). The WarmStart property enables high throughput applications, room temperature setup, and increases the consistency and specificity of amplification reactions. RTx contains intact RNase H activity.

WarmStart RTx Reverse Transcriptase (Glycerol-free) offers the same robust detection of human RNA targets in RT-LAMP assays as the glycerol-containing enzyme.

WarmStart® RTx Reverse Transcriptase (Glycerol-free) |

RT-LAMP (RNA targets) experiments were performed with NEB #M0380: WarmStart RTx Reverse Transcriptase and NEB #M0538: Bst 2.0 WarmStart DNA Polymerase, or NEB #M0439: WarmStart RTx Reverse Transcriptase (Glycerol-free) and NEB #M0402: Bst 2.0 WarmStart DNA Polymerase (Glycerol-free). Reactions containing 1X LAMP primers and 0.5X LAMP Fluorescent dye were set up in quadruplicate over three logs of total Jurkat RNA (10 ng to 0.1 ng) in 96-well, 25 µl reactions. Control reactions without template (NTC) were also evaluated. Reactions were incubated at 65°C for 40 minutes and fluorescence was monitored every 15 seconds in the SYBR/FAM channel of a real-time thermocycler (Bio-Rad® CFX96). Each dot represents the time at which the fluorescence signal for a single reaction crosses the instrument-defined threshold. All four replicates were detected at each template input unless otherwise indicated (note that dots frequently overlap given similar detection time for the replicates). Overall, similar performance was observed for both glycerol-containing (NEB #M0538 and NEB #M0380) and glycerol-free (NEB #M0402: and NEB #M0439) enzymes at each template input. No amplification was observed in any of the no template control reactions.

产品类别:
Isothermal Amplification & Strand Displacement Products,
cDNA Synthesis & Reverse Transcriptases Products,
PCR, qPCR & Amplification Technologies Products

应用:
Loop-Mediated Isothermal Amplification,
DNA Amplification, PCR & qPCR

  • 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
    • M0439L     -80    
        WarmStart® RTx Reverse Transcriptase (Glycerol-free) M0439LVIAL -80 1 x 0.027 ml 75,000 units/ml
        10X Isothermal Amplification Buffer (Lyo-compatible) B1714SVIAL -20 1 x 1.25 ml 10 X

操作说明、说明书 & 用法

  • 操作说明

    1. Typical RT-LAMP Protocol (NEB #M0439)
    2. Typical cDNA Synthesis Protocol with WarmStart® RTx Reverse Transcriptase (Glycerol-free) (NEB #M0439)

工具 & 资源

  • Web 工具

    • NEB LAMP Primer Design Tool

FAQs & 问题解决指南

  • FAQs

    1. What is LAMP and RT-LAMP?
    2. What advantages does WarmStart® RTx provide?
    3. How sensitive is RNA amplification with WarmStart® RTx?
    4. Can WarmStart® RTx be inactivated?
    5. Is WarmStart® RTx active in other buffers?
    6. What is the maximum length of cDNA product produced by WarmStart® RTx?
    7. Does WarmStart® RTx have RNase H activity?
    8. Can DNA be used as a template for WarmStart® RTx?
    9. What are Hot Start and WarmStart® polymerases and when would I use them?
    10. What is the difference between buffers B1714: 10X Isothermal Amplification Buffer (Lyo-compatible) and B0537: 10X Isothermal Amplification Buffer?
    11. What is the Mg2+ concentration in the 10X Isothermal Amplification Buffer (Lyo-compatible)?
    12. Does B1714: 10X Isothermal Amplification Buffer (Lyo-compatible) include any excipients?
    13. Can WarmStart® RTx Reverse Transcriptase (Glycerol-free) at 75,000 U/mL be diluted prior to use?
    14. How many Freeze/Thaw cycles can WarmStart® RTx Reverse Transcriptase (Glycerol-free) tolerate?

质控、安全 & 法规

  • 质控分析

    每一新批次的 NEB 产品都要经过质控，以满足指定的质量标准，对特定产品的质量标准和每一批次的检测数据可以通过产品说明表格、COA、产品信息卡或者产品手册进行查阅或下载。关于 NEB 产品质控的详细信息，可从此处查阅。

  • 产品说明与变更通知

    产品说明表包含产品的储存温度、有效期和规格，这些文件的命名规则如下：[货号]_[规格]_[版本]

    • M0439L_v1

  • CoA 文件包含单个批次产品的储存温度、有效期和质控，这些文件的命名规则如下： [货号]_[规格]_[版本]_[Lot#]

    • M0439L_v1_10225699

HIV-1 Reverse Transcriptase, Recombinant

发表于由

HIV-1 Reverse Transcriptase, Recombinant

品牌：Bio Academia
CAS No.：
储存条件：-20℃
纯度：>90%
产品编号

(生产商编号)

 等级 规格 运输包装 零售价(RMB) 库存情况 参考值

05-001

 – 200 units – 咨询 – –


* 干冰运输、大包装及大批量的产品需酌情添加运输费用


* 零售价、促销产品折扣、运输费用、库存情况、产品及包装规格可能因各种原因有所变动，恕不另行通知，确切详情请联系上海金畔生物科技有限公司。

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?

WarmStart® RTx 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

WarmStart® RTx Reverse Transcriptase |

WarmStart RTx Reverse Transcriptase is a unique in silico designed RNA-directed DNA polymerase coupled with a reversibly-bound aptamer that inhibits RTx activity below 40°C. This enzyme can synthesize a complementary DNA strand initiating from a primer using RNA (cDNA synthesis) or single-stranded DNA as a template. RTx is a robust enzyme for RNA detection in amplification reactions and is particularly well suited for use in LAMP (loop-mediated isothermal amplification). The WarmStart property enables high throughput applications, room temperature setup, and increases the consistency and specificity of amplification reactions. RTx contains intact RNase H activity.

To support lyophilization, incorporation into microfluidic devices, and enable quick adoption into automation workflows, WarmStart RTx Reverse Transcriptase is also offered in a glycerol-free format.

Improved Speed and Sensitivity in RT-LAMPWarmStart® RTx Reverse Transcriptase |
RT-LAMP reactions with Bst 2.0 WarmStart DNA Polymerase and the indicated reverse transcriptase were incubated at 65°C with 1 pg – 100 ng of Jurkat total RNA. Reactions were monitored with real-time fluorescence, and the resulting curves are shown on left, with the corresponding threshold times on right. WarmStart RTx provides faster reaction threshold times for improved consistancy and sensitivity with lower input RNA amounts. RT-LAMP reactions performed with AMV Reverse Transcriptase resulted in inconsistent detection, as indicated by wide variation at lower RNA input concentrations (blue curves).
Sensitive Detection with Reduced False Negatives

Total RNA WarmStart RTx AMV RT
100 ng 3/3 3/3
10 ng 3/3 3/3
1 ng 3/3 3/3
100 pg 3/3 2/3
10 pg 3/3 2/3
1pg 3/3 1/3
RT-LAMP reactions were performed with WarmStart RTx and AMV Reverse Transcriptase with total RNA at 6 input concentrations. Positive detection, indicated by fluorescent detection with a twenty-minute reaction time, was achieved down to 1 pg of total input RNA. Use of AMV resulted in false negatives when total RNA was 100 pg or lower.
WarmStart Control of WarmStart RTxWarmStart® RTx Reverse Transcriptase |
cDNA synthesis was performed for 10 minutes followed by qPCR analysis. Resulting Cts were normalized against both a “no RT” control for 0% activity and the fastest Ct for 100% activity. WarmStart RTx is inhibited by a reversibly-bound aptamer at temperatures below 40°C.
Robust RT-LAMP Across a Range of TargetsWarmStart® RTx Reverse Transcriptase |
A variety of RT-LAMP targets were amplified using Bst 2.0 WarmStart DNA Polymerase and the indicated reverse transcriptase. Reactions with WarmStart RTx resulted in faster threshold times and more consistent reaction success across a range of targets and copy number. 
产品类别:
RT-PCR Products,
cDNA Synthesis & Reverse Transcriptases Products

应用:
Strand Displacement Amplification & Nicking Enzyme,
cDNA Synthesis,
Reverse Transcription (cDNA Synthesis),

Loop-Mediated Isothermal Amplification,
RT-PCR & cDNA Synthesis,
Isothermal Amplification,

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
    • M0380S     -20    
        WarmStart® RTx Reverse Transcriptase M0380SVIAL -20 1 x 0.027 ml 15,000 units/ml
        Isothermal Amplification Buffer Pack B0537SVIAL -20 1 x 1.5 ml 10 X
        Magnesium Sulfate (MgSO4) Solution B1003SVIAL -20 1 x 1.5 ml 100 mM
    • M0380L     -20    
        WarmStart® RTx Reverse Transcriptase M0380LVIAL -20 1 x 0.134 ml 15,000 units/ml
        Isothermal Amplification Buffer Pack B0537SVIAL -20 1 x 1.5 ml 10 X
        Magnesium Sulfate (MgSO4) Solution B1003SVIAL -20 1 x 1.5 ml 100 mM

  • 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 20 minutes at 50°C using poly(rA)•oligo(dT)18 as template.

    反应条件

    1X Isothermal Amplification Buffer Pack

    1X Isothermal Amplification Buffer Pack
    20 mM Tris-HCl
    10 mM (NH4)2SO4
    50 mM KCl
    2 mM MgSO4
    0.1% Tween® 20
    (pH 8.8 @ 25°C)

    贮存溶液

    100 mM KCl
    10 mM Tris-HCl
    0.1 mM EDTA
    1 mM DTT
    50% Glycerol
    pH 7.4 @ 25°C

    热失活

    80°C for 10 minutes

    单位活性检测条件

    20 mM Tris-HCl (pH 8.8), 10 mM (NH4)2SO4, 50 mM KCl, 2 mM MgSO4, 0.1% Tween-20, 0.5 mM dTTP, 0.4 mM poly(rA)•oligo(dT)18.

  • 优势和特性

    应用特性

    • RT-LAMP
    • cDNA synthesis
    • RT reactions requiring room temperature setup

  • 相关产品

    相关产品

    • 随机引物混合液
    • 小鼠 RNase 抑制剂
    • dNTP 套装
    • dNTP 混合液
    • m0537-bst-20-dna-polymerase
    • m0538-bst-20-warmstart-dna-polymerase
    • 等温扩增缓冲液套装

  • 注意事项

    1. 反应条件：25 µl 反应体系：1X 等温扩增反应缓冲液、模板、引物、dNTP 和 0.25 – 0.5 µl WarmStart RTx 反转录酶。50℃ – 55℃ 温育进行 cDNA 合成；或直接 65℃ 反应进行一步法 RT-LAMP。

操作说明、说明书 & 用法

  • 操作说明

    1. Typical RT-LAMP Protocol
    2. Typical cDNA Synthesis Protocol with WarmStart RTx (NEB #M0380)

工具 & 资源

  • Web 工具

    • NEB LAMP Primer Design Tool

FAQs & 问题解决指南

  • FAQs

    1. The product size was previously listed in Reactions and is now listed in Units. What has changed?
    2. What is LAMP and RT-LAMP?
    3. What advantages does WarmStart® RTx provide?
    4. How sensitive is RNA amplification with WarmStart® RTx?
    5. Can WarmStart® RTx be inactivated?
    6. Is WarmStart® RTx active in other buffers?
    7. How do I use WarmStart® RTx in RT-LAMP?
    8. What is the maximum length of cDNA product produced by WarmStart® RTx?
    9. How do I use WarmStart® RTx for cDNA synthesis?
    10. Does WarmStart® RTx have RNase H activity?
    11. What dNTPs do you recommend for use with WarmStart® RTx?
    12. Can DNA be used as a template for WarmStart® RTx?
    13. Does NEB have a master mix for LAMP or RT-LAMP reactions?