LunaScript RT Master Mix Kit Protocols (NEB #E3025)

  1. Mix components by vortexing briefly and spin down if necessary.

  2. Prepare cDNA synthesis reaction as described below:

    COMPONENT 20 µl REACTION FINAL CONCENTRATION
    LunaScript RT Master Mix (Primer-free) (5X) 4 µl

    1X

    Random Primer Mix (60 µM)1
    Or d(T)23VN (50 µM) 2
    Or Gene-specific primer3
    2 μl
    2 μl 
    x μl 
    6 mM
    5 mM
    0.1–1 µM (typically 0.5 µM final)
    RNA Sample4 variable

    (up to 1 µg)

    Nuclease-free Water to 20 µl

    For no-RT control reactions, mix the following components:

    COMPONENT 20 µl REACTION FINAL CONCENTRATION
    No-RT Control Mix (Primer-free) (5X) 4 µl

    1X

    Random Primer Mix (60 µM)1
    Or d(T)23VN (50 µM) 2
    Or Gene-specific primer3
    2 μl 
    2 μl 
    x μl 
    6 mM
    5 mM
    0.1–1 µM (typically 0.5 µM final)
    RNA Sample4 variable

    (up to 1 µg)

    Nuclease-free Water to 20 µl

    For no template controls, mix the following components:

    COMPONENT 20 µl REACTION FINAL CONCENTRATION
    LunaScript RT Master Mix (Primer-free) (5X) 4 µl

    1X

    Random Primer Mix (60 µM)1
    Or d(T)23VN (50 µM) 2
    Or Gene-specific primer3
    2 μl 
    2 μl 
    x μl 
    6 mM
    5 mM
    0.1–1 µM (typically 0.5 µM final)
    Nuclease-free Water to 20 µl

    1 Random Primer Mix (NEB #S1330) is recommended for real-time qPCR detection.
    2 Oligo d(T)23VN (NEB #S1327) is recommended for long or full-length cDNA synthesis.
    3 Gene-specific primers can be used for target-specific cDNA synthesis. The final concentration is typically 0.5 µM and can be optimized in the range of 0.1–1 µM.
    4 Up to 1 µg total RNA, 1 µg mRNA or 100 ng specific RNA can be used in a 20 µl reaction. To accommodate larger amounts of input RNA (> 1 µg), the reaction should be scaled up to ensure optimum cDNA synthesis.

    Incubate reactions in a thermocycler with the following steps:

    PRIMERS FOR cDNA SYNTHESIS

    CYCLE STEP

    TEMP

    TIME CYCLES
    Random Primers (e.g., Random Primer Mix)

    Primer Annealing

    25°C

    2 minutes

    1

    cDNA Synthesis

    55°C

    10 minutes

    Heat Inactivation

    95°C

    1 minute
    Oligo-dT primers or a gene-specific primer

    cDNA Synthesis

    55°C

    10 minutes

    1

    Heat Inactivation

    95°C

    1 minute


The cDNA product should be stored at -20°C. In general, the volume of cDNA product should not exceed 1/10 of the qPCR or PCR reaction volume. Where needed, up to 20% qPCR volume can be undiluted cDNA product.

For qPCR applications, we recommend using Luna Universal qPCR Master Mix (NEB #M3003) for dye-based qPCR detection and Luna Universal Probe qPCR Master Mix (NEB #M3004) for probe-based detection.

For downstream PCR, we recommend OneTaq 2X Master Mix (NEB #M0482 or NEB #M0485) for PCR detection up to 5kb, Q5® Hot Start High-Fidelity 2X Master Mix (NEB #M0494) for highest fidelity, and LongAmp Taq 2X Master Mix (NEB #M0287) for high yields from longer products.