Protocol for LunaScript® RT SuperMix Kit (E3010)
- Mix components briefly and spin down if necessary.
- Prepare cDNA synthesis reaction as described below:
COMPONENTS 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
For no-RT control reaction, mix the following components:
COMPONENTS 20 μl REACTION FINAL CONCENTRATION No-RT Control Mix (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:
COMPONENTS 20 μl REACTION FINAL CONCENTRATION LunaScript RT SuperMix (5X)
4 µl 1X
Nuclease-free Water
to 20 µl
Incubate reactions in a thermocycler with the following steps:
CYCLE STEP TEMPERATURE TIME CYCLES Primer Annealing
25°C
2 minutes
1 cDNA Synthesis
55°C
10 minutes Heat Inactivation 95°C
1 minute