• My NEB
  • Print
  • PDF
  • OneTaq® RT-PCR Kit

    Description

    OneTaq® RT-PCR Kit combines two powerful mixes, M-MuLV Enzyme Mix and OneTaq Hot Start 2X Master Mix with Standard Buffer for 2-step RT-PCR applications. The two mixes require minimal handling during reaction setup and yet offer consistent and robust RT-PCR reactions.

    The first strand cDNA synthesis is achieved by using two optimized mixes, M-MuLV Enzyme Mix and M-MuLV Reaction Mix. M-MuLV Enzyme Mix combines M-MuLV Reverse Transcriptase and murine RNase Inhibitor while M-MuLV Reaction Mix contains dNTPs and an optimized buffer. The kit also contains 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 amplification step features a OneTaq Hot Start DNA Polymerase in a master mix format. OneTaq Hot Start DNA Polymerase offers higher fidelity than Taq and better amplification. RT-PCR product up to 6 kb can be generated (Figure 1).

    Figure 1. First Strand DNA Synthesis
    First strand cDNA systhesis was carried out in the presence of 1X M-MuLV Enzyme Mix at 42°C using 0.5 μg of human spleen total RNA in the presence of dT23VN (lanes 1, 4 and 7) or Random Hexamer Mix (lanes 2, 5 and 8). No-RT controls were lanes 3, 6 and 9. OneTaq Hot Start 1X Master Mix was used to amplify a 1.5 kb fragment of beta-actin gene, a 0.6 kb fragment of GAPDH gene, and a 5.5 kb fragment from p532 gene in 35 cycles. The marker lane (M) contains 2-Log DNA Ladder (NEB #N3200 ).
    Figure 2. First Strand DNA Synthesis

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Oligo d(T)23 VN-2050 μM
    Nuclease-free Water
    M-MuLV Enzyme Mix10X
    M-MuLV Reaction Mix2X
    Random Primer Mix-2060 μM
    OneTaq Hot Start 2X Master Mix with Standard Buffer2X

    Quality Control

    Quality Assurance Statement

    • The performance of OneTaq RT-PCR Kit is tested in an RT reaction using human Jurkat total RNA with primer d(T)23VN. The sensitivity of the kit is verified by the detection of GAPDH transcript in 20 pg total RNA after 35 cycles. The length of cDNA achieved is verified by the detection of a 5.5 kb amplicon of the p532 gene.

    References

    1. Liao, J. and Gong, Z. (1997). Biotechniques. 23, 368-370.
    2. Van Gilst, M.R. et al. (2005). PLoS Biology. 3, 301-312.
    3. Sambrook, J. and Russel, D.W. (2001). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press. 3rd Ed..
    4. Don, R.H. et al. (1991). Nucleic Acid Research. 19, 4008.
    5. Aguila et al. (2005). BMC Molecular Biology. 6, 9.

    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. Why am I getting a low yield of cDNA?
    2. Why do I see products of the wrong size?
    3. Why do I have a low yield of PCR product?
    1. PCR Amplification with OneTaq® RT-PCR Kit
    2. First strand cDNA synthesis OneTaq® RT-PCR Kit