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  • ProtoScript® II First Strand cDNA Synthesis Kit

    Description

    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.

    E6560
    Figure 1: cDNA Synthesis of Jurkat RNA with the ProtoScript II First Strand cDNA Synthesis Kit
    First strand cDNA systhesis 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
    2-Log DNA Ladder (NEB #N3200).
    Quantitative detection of reverse transcription products using the ProtoScript II First Strand cDNA Synthesis Kit
    Ten-fold serial dilutions of in vitro transcribed luciferase RNA (1x102 to 1x109 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.

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Oligo d(T)23 VN-2050 μM
    Random Primer Mix-2060 μM
    Nuclease-free Water
    ProtoScript® II Reaction Mix-202X
    ProtoScript® II Enzyme Mix-2010X

    Properties and Usage

    Storage Temperature

    -20°C

    Troubleshooting

    Troubleshooting

    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.

    References

    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.

    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.

    Manuals

    The Product Manual 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 these document files: manual[Catalog Number].
    1. What is the difference between E6560 and E6300?
    2. What is the reaction temperature for E6560?
    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. Is RNaseH treatment required before PCR amplification?
    1. First Strand cDNA Synthesis Protocols (E6560)

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