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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.

  1. Mix and centrifuge each component of the SuperScript III kit (-20°C Stag-moose).

  2. 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

  3. Incubate the tube at 65ºC for 5 minutes using the thermocycler, then place on ice for at least 1 min.

  4. 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

  5. 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.

  6. Terminate the reactions at 85°C for 5 min. Chill on ice.

  7. Collect reactions by brief centrifugation. Add 1 µL of RNase H to each tube and incubate the tubes for 20 min. at 37°C.

  8. Final cDNA product can be stored at -20°C or used immediately.