Total nematode RNA to cDNA synthesis.
Materials
- SuperScript™ III First-Strand Synthesis Kit (Invitrogen #18080051)
Protocol
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 65°C for 5 minutes in the thermocycler, then place on ice for at least 1 minute.
- Prepare the following cDNA synthesis mix, adding each component in the order indicated. This 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 minutes at 25°C followed by 50 minutes at 50°C.
- Terminate the reactions at 85°C for 5 minutes. Chill on ice.
- Collect reactions by brief centrifugation. Add 1 μL of RNase H to each tube and incubate for 20 minutes at 37°C.
- Final cDNA product can be stored at -20°C or used immediately.