标签归档：protoscript

ProtoScript® cDNA 第一链合成试剂盒 |NEB酶试剂 New England Biolabs

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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® cDNA 第一链合成试剂盒含两种经优化的混合液：M-MuLV 酶混合液和 M-MuLV 反应混合液。M-MuLV 酶混合液含 M-MuLV 反转录酶和小鼠 RNase 抑制剂，M-MuLV 反应混合液含 dNTP 和经优化的缓冲液。该试剂盒还包含两种优化的反转录引物和无核酸酶水。锚定的 Oligo-dT 引物 [d(T)23VN] 迫使引物与 polyA 尾的起始端退火。经优化的随机引物混合液能随机并持续性地与整个 RNA 模板配对，包括 mRNA 和无 polyA 尾的 RNA。合成的第一链 cDNA 产物长度可超过 10 kb（图 1）。

成功合成 cDNA 的要素：

RNA 模板
高纯度的完整 RNA 对于灵敏的 RT-PCR 检测至关重要。RNA 的 A260/A280 比值应至少 1.7 或更高。

总 RNA 或 mRNA 均可用于反转录反应。总 RNA 通常足以进行大多数 RT-PCR 分析。不过，如果需要，可以使用 PolyA Spin mRNA 分离试剂盒（NEB #S1560）或 mRNA 磁性分离试剂盒（NEB #S1550）获得 mRNA。

检测所需的 RNA 量取决于目标转录本的丰度。通常推荐使用 1 ng 至 1 μg 的总 RNA 或 0.1-100 ng 的 mRNA。

cDNA 第一链合成反应
RNA 和引物在 70℃ 条件下变性 5 分钟，可以去除阻碍较长 cDNA 合成的二级结构。不过，可以在某些情况下省略此步骤（结果未发表）。

推荐在 42℃ 条件下温育一小时，以获得最高产量和最大长度。不过，许多靶标用较短的温育时间即可进行检测。例如，对于 2 kb cDNA 合成，温育 5 分钟就足够了。

选择反转录引物
Oligo d(T) 引物是大多数实验的首选，因为它确保所有 cDNA 拷贝在 mRNA 的 3´ 端终止并产生最长的连续 cDNA。锚定的 Oligo-dT 引物 [d(T)23VN] 迫使引物与 polyA 尾的起始端退火，从而防止在 polyA 尾的内部位点开始退火（1）。不过，如果需要，也可以选择另外两种引物。

随机引物混合液是经优化的六聚体和 d(T)23VN 引物混合液。该混合液能随机地与整个 RNA 模板配对，包括 mRNA 和无 polyA 尾的 RNA（例如核糖体 RNA）。随机引物混合液产生的 cDNA 平均长度较短，可用于检测多个短 RT-PCR 产物。随机引物混合液在各种 RNA 模板中均具有良好的性能。

基因特异引物用于 cDNA 合成反应时，cDNA 产物只能用于扩增该转录本。RNA 起始量较低（低于 10 ng）且只需要一种特定的 cDNA 时，使用这种方法可以获得良好的结果。

推荐的引物浓度：
引物：终浓度
OLIGO d(T)23VN：5 μM
随机引物混合液：6 μM
特异引物：0.1–1 μM

图 1： ProtoScript® cDNA 第一链合成试剂盒 |
以 2 μg 人脾总 RNA 为模板，在 42℃ 条件下，使用 1X M-MuLV 酶混合液合成 cDNA 第一链。阴性对照反应使用 1X M-MuLV 反应混合液进行。以部分 cDNA 第一链产物为模板，使用 1X LongAmp™ Taq 2X 预混液（NEB #M0287）扩增三种不同 mRNA 的特异序列。泳道 1：β 肌动蛋白基因的 1.1 kb 片段。泳道 2：β 肌动蛋白基因 1.1 kb 片段的 noRT 对照。泳道 3：Xrn-1 基因的 4.7 kb 片段。泳道 4：Xrn-1 基因 4.7 kb 片段的 noRT 对照。泳道 5：鸟嘌呤核苷酸交换因子 p532 的 9.8 kb 片段。泳道 6：鸟嘌呤核苷酸交换因子 p532 9.8 kb 片段的 noRT 对照。Marker M 为 1 kb Plus DNA Ladder（NEB #N3200）。
产品类别:
RT-PCR Products,
cDNA Synthesis & Reverse Transcriptases Products

应用:
cDNA Synthesis,
Reverse Transcription (cDNA Synthesis),
RT-PCR & cDNA Synthesis,

PCR

  • 试剂盒组成

    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
    • E6300S     -20    
        M-MuLV Enzyme Mix M0342AVIAL -20 1 x 0.06 ml 10 X
        M-MuLV Reaction Mix M0343AVIAL -20 1 x 0.4 ml 2 X
        Random Primer Mix S1330AVIAL -20 1 x 0.07 ml 60 µM
        Oligo d(T)23 VN S1332AVIAL -20 1 x 0.07 ml 50 µM
        Nuclease-free Water B1502AVIAL -20 1 x 1.5 ml Not Applicable
    • E6300L     -20    
        M-MuLV Enzyme Mix M0342AVIAL -20 5 x 0.06 ml 10 X
        M-MuLV Reaction Mix M0343AVIAL -20 5 x 0.4 ml 2 X
        Random Primer Mix S1330AVIAL -20 5 x 0.07 ml 60 µM
        Oligo d(T)23 VN S1332AVIAL -20 5 x 0.07 ml 50 µM
        Nuclease-free Water B1502AVIAL -20 5 x 1.5 ml Not Applicable

  • 相关产品

    相关产品

    • s1550-magnetic-mrna-isolation-kit
    • 随机引物混合液
    • NEBNext® Ultra II RNA 非链特异性第二链合成模块
    • Oligo d(T)25 磁珠
    • LongAmp® Taq 2X 预混液
    • Taq 2X 预混液

  • 参考文献

    1. Liao, J. and Greg, Z. (1997). Biotechniques. 23, 368-370.
    2. Sambrook, J. and Russel, D.W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). (pp. 8.46-8.53 and 11.37-11.42). Cold Spring Harbor.

操作说明、说明书 & 用法

  • 操作说明

    1. First Strand cDNA Synthesis Protocols (E6300)

  • 说明书

    产品说明书包含产品使用的详细信息、产品配方和质控分析。

    • manualE6300

  • 使用指南

    • cDNA/Reverse Transcriptase Tips

FAQs & 问题解决指南

  • FAQs

    1. How do I choose between LunaScript RT SuperMix Kit and ProtoScript first strand cDNA synthesis kits?
    2. Why am I getting a low yield of cDNA?

ProtoScript® II First Strand cDNA Synthesis Kit |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 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 KitProtoScript® II First Strand cDNA Synthesis Kit |
First strand cDNA synthesis 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
the 1 kb Plus DNA Ladder (NEB #N3200).
Quantitative detection of reverse transcription products using the ProtoScript II First Strand cDNA Synthesis KitProtoScript® II First Strand cDNA Synthesis Kit |
Ten-fold serial dilutions of in vitro transcribed luciferase RNA (1×102 to 1×109 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.
产品类别:
RT-PCR Products,
cDNA Synthesis & Reverse Transcriptases Products

应用:
cDNA Synthesis,
Reverse Transcription (cDNA Synthesis),
RT-PCR & cDNA Synthesis,

PCR

  • 试剂盒组成

    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
    • E6560S     -20    
        ProtoScript® II Reaction Mix M0555AVIAL -20 1 x 0.4 ml 2 X
        ProtoScript® II Enzyme Mix M0556AVIAL -20 1 x 0.06 ml 10 X
        Random Primer Mix S1330AVIAL -20 1 x 0.07 ml 60 µM
        Nuclease-free Water B1502AVIAL -20 1 x 1.5 ml Not Applicable
        Oligo d(T)23 VN S1332AVIAL -20 1 x 0.07 ml 50 µM
    • E6560L     -20    
        ProtoScript® II Reaction Mix M0555AVIAL -20 5 x 0.4 ml 2 X
        ProtoScript® II Enzyme Mix M0556AVIAL -20 5 x 0.06 ml 10 X
        Random Primer Mix S1330AVIAL -20 5 x 0.07 ml 60 µM
        Nuclease-free Water B1502AVIAL -20 5 x 1.5 ml Not Applicable
        Oligo d(T)23 VN S1332AVIAL -20 5 x 0.07 ml 50 µM

  • 相关产品

    相关产品

    • s1550-magnetic-mrna-isolation-kit
    • Oligo d(T)25 磁珠
    • 小鼠 RNase 抑制剂
    • 随机引物混合液
    • LongAmp® Taq 2X 预混液
    • Taq 2X 预混液

  • 参考文献

    1. Liao, J. and Gong, Z. (1997). Biotechniques. 23, 368-370.
    2. Sambrook, J. and Russel, D.W. (2001). Molecular Cloning: A Laboratory Manual, (3rd ed.),. (pp. 8.46–8.53 avnd 11.37–11.42). Cold Spring Harbor: Cold Spring Harbor Laboratory Press.

操作说明、说明书 & 用法

  • 操作说明

    1. First Strand cDNA Synthesis Protocols (E6560)

  • 说明书

    产品说明书包含产品使用的详细信息、产品配方和质控分析。

    • manualE6560

  • 使用指南

    • cDNA/Reverse Transcriptase Tips

FAQs & 问题解决指南

  • FAQs

    1. How do I choose between LunaScript RT SuperMix Kit and ProtoScript first strand cDNA synthesis kits?
    2. What is the difference between E6560 and E6300?
    3. What is the reaction temperature for E6560?
    4. Is RNaseH treatment required before PCR amplification?
    5. Can the cDNA products be used in real-time PCR analysis?
    6. What thermostable DNA polymerase can be used for PCR after cDNA synthesis?
    7. How do I choose between Induro Reverse Transcriptase and the different LunaScript and ProtoScript products?
    8. Why do I have low cDNA yields?
    9. How do I know whether my template RNA is of good quality?

  • 问题解决指南

    Problem Suggestion
    Low Yield of cDNA Check the integrity of the RNA by denaturing agarose gel electrophoresis (2). RNA should have a minimum A260/A280 ratio of 1.7 or higher. Ethanol precipitation followed by a 70% ethanol wash can remove most contaminants such as EDTA and guanidinium. Precipitation with lithium chloride can remove polysaccharides (2).
      Phenol/chloroform extraction and ethanol extraction can remove contaminant proteins such as proteases (2).
      Some target RNA may contain strong pauses for RT; Use random priming instead of d(T)23VN.
      Use sufficient amount of RNA.

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?