LunaScript RT SuperMix Protocol (NEB #M3010)
- Mix components briefly and spin down if necessary.
- 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