Total Nematode RNA to cDNA Synthesis
Materials
- SuperScript™ III First-Strand Synthesis Kit (Invitrogen #18080051)
Protocol
Note: Follow the kit protocol, with minor modifications that include the mixing of Oligo dT and Random Hexamer primers.
-
Mix and centrifuge each component of the SuperScript III kit (-20°C Stag-moose).
-
Combine the following reaction in a 0.2 mL PCR tube. For cloning, maximize the total RNA input (8 µL). For qPCR, standardize total RNA input across samples.
Component Volume Total Parasite RNA n µL Oligo dT 1 µL Random Hexamer 1 µL 10 mM dNTP mix 1 µL Molecular Grade H2O to 10 µL -
Incubate the tube at 65ºC for 5 minutes using the thermocycler, then place on ice for at least 1 min.
-
Prepare the following cDNA Synthesis Mix, adding each component in the indicated order. Synthesis Mix is enough for one reaction (scale accordingly).
Component Volume 10X RT Buffer 2 µL 25 mM MgCl2 4 µL 0.1 M DTT 2 µL RNaseOUT (40 U/µL) 1 µL SuperScript III RT (200 U/µL) 1 µL TOTAL 10 µL -
Add 10 µL of cDNA Synthesis Mix to each RNA/primer mix, mix gently, collect by centrifugation and incubate for 10 min. at 25°C, followed by 50 min. at 50°C.
-
Terminate the reactions at 85°C for 5 min. Chill on ice.
-
Collect reactions by brief centrifugation. Add 1 µL of RNase H to each tube and incubate the tubes for 20 min. at 37°C.
-
Final cDNA product can be stored at -20°C or used immediately.