Home Protocols LunaScript RT SuperMix Protocol (NEB #M3010)

LunaScript RT SuperMix Protocol (NEB #M3010)

  1. Mix components briefly and spin down if necessary.

  2. Prepare cDNA synthesis reaction as described below:

    COMPONENT 20 µl REACTION FINAL CONCENTRATION
    LunaScript RT SuperMix (5X) 4 µl 1X
    RNA Sample variable (up to 1 μg)*
    Nuclease-free Water to 20 µl

    *Up to 1 µg total RNA, 1 µg mRNA or 100 ng specific RNA can be used in a 20 µl reaction.
    However, the cDNA input for downstream qPCR detection should typically contain < 109 copies of the target to ensure that quantitation remains linear. To accommodate larger amounts of input RNA (> 1 µg), the reaction should be scaled up to ensure optimum cDNA synthesis.

    For no template controls, mix the following components:

    COMPONENT 20 µl REACTION FINAL CONCENTRATION
    LunaScript RT SuperMix (5X) 4 µl 1X
    Nuclease-free Water 16 µl  

    Incubate reactions in a thermocycler with the following steps:

    CYCLE STEP TEMP TIME CYCLES
    Primer Annealing 25°C 2 minutes 1
    cDNA Synthesis 55°C 10 minutes
    Heat Inactivation 95°C 1 minute