ProtoScript® II First Strand cDNA Synthesis Kit |NEB酶试剂 New England Biolabs

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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.